scholarly journals Lactobacillus buchneri Genotyping on the Basis of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Locus Diversity

2013 ◽  
Vol 80 (3) ◽  
pp. 994-1001 ◽  
Author(s):  
Alexandra E. Briner ◽  
Rodolphe Barrangou
Keyword(s):  
Immune System ◽  
Comparative Analysis ◽  
Common Origin ◽  
Type Ii ◽  
Crispr Locus ◽  
Content Type ◽  
Lactobacillus Buchneri ◽  
Protospacer Adjacent Motif ◽  
Associated Sequences ◽  
Short Palindromic Repeat

ABSTRACTClustered regularly interspaced short palindromic repeats (CRISPR) in combination with associated sequences (cas) constitute the CRISPR-Cas immune system, which uptakes DNA from invasive genetic elements as novel “spacers” that provide a genetic record of immunization events. We investigated the potential of CRISPR-based genotyping ofLactobacillus buchneri, a species relevant for commercial silage, bioethanol, and vegetable fermentations. Upon investigating the occurrence and diversity of CRISPR-Cas systems inLactobacillus buchnerigenomes, we observed a ubiquitous occurrence of CRISPR arrays containing a 36-nucleotide (nt) type II-A CRISPR locus adjacent to fourcasgenes, including the universalcas1andcas2genes and the type II signature genecas9. Comparative analysis of CRISPR spacer content in 26L. buchneripickle fermentation isolates associated with spoilage revealed 10 unique locus genotypes that contained between 9 and 29 variable spacers. We observed a set of conserved spacers at the ancestral end, reflecting a common origin, as well as leader-end polymorphisms, reflecting recent divergence. Some of these spacers showed perfect identity with phage sequences, and many spacers showed homology toLactobacillusplasmid sequences. Following a comparative analysis of sequences immediately flanking protospacers that matched CRISPR spacers, we identified a novel putative protospacer-adjacent motif (PAM), 5′-AAAA-3′. Overall, these findings suggest that type II-A CRISPR-Cas systems are valuable for genotyping ofL. buchneri.

scholarly journals Primed CRISPR-Cas Adaptation and Impaired Phage Adsorption in Streptococcus mutans

mSphere ◽  
2021 ◽  
Vol 6 (3) ◽  
Author(s):  
Cas Mosterd ◽  
Sylvain Moineau
Keyword(s):  
Streptococcus Mutans ◽  
Defense Mechanisms ◽  
Phage Genome ◽  
Multiplicity Of Infection ◽  
Type Ii ◽  
Content Type ◽  
Virulent Phage ◽  
Phage Adsorption ◽  
Protospacer Adjacent Motif

ABSTRACT Streptococcus mutans strain P42S possesses a type II-A CRISPR-Cas system that protects against phage infection and plasmid transformation. The analysis of 293 bacteriophage-insensitive mutants (BIMs) obtained upon exposure to the virulent phage M102AD revealed the acquisition of 399 unique spacers, including several ectopic spacer acquisitions and a few cases of native spacer deletions. The acquisition of multiple spacers was also observed and appears to be mostly due to priming, which has been rarely reported for type II-A systems. Analyses of the acquired spacers indicated that 88% of them are identical to a region of the phage M102AD genome. The remaining 12% of spacers had mismatches with the phage genome, primarily at the 5′ end of the spacer, leaving the seed sequence at the 3′ end largely intact. When a high multiplicity of infection (MOI) was used in the phage challenge assays, we also observed the emergence of CRISPR BIMs that, in addition to the acquisition of new spacers, displayed a reduced phage adsorption phenotype. While CRISPR-Cas and adsorption resistance work in tandem to protect S. mutans P42S against phage M102AD, nonidentified antiviral mechanisms are also likely at play in this strain. IMPORTANCE Bacteria are under the constant threat of viral predation and have therefore developed several defense mechanisms, including CRISPR-Cas systems. While studies on the mode of action of CRISPR-Cas systems have already provided great insights into phage-bacterium interactions, still more information is needed on the biology of these systems. The additional characterization of the type II-A CRISPR-Cas system of Streptococcus mutans P42S in this study provides novel information on the spacer acquisition step, especially regarding protospacer-adjacent motif (PAM) recognition, multiple-spacer acquisition, and priming.

scholarly journals Impact of Different Target Sequences on Type III CRISPR-Cas Immunity

2016 ◽  
Vol 198 (6) ◽  
pp. 941-950 ◽  
Author(s):  
Inbal Maniv ◽  
Wenyan Jiang ◽  
David Bikard ◽  
Luciano A. Marraffini
Keyword(s):  
Immune System ◽  
Adaptive Immune System ◽  
Arms Race ◽  
Conjugative Plasmid ◽  
Target Sequence ◽  
Sequence Matching ◽  
Content Type ◽  
Genetic Elements ◽  
Crispr System ◽  
Short Palindromic Repeat

ABSTRACTClustered regularly interspaced short palindromic repeat (CRISPR) loci encode an adaptive immune system of prokaryotes. Within these loci, sequences intercalated between repeats known as “spacers” specify the targets of CRISPR immunity. The majority of spacers match sequences present in phages and plasmids; however, it is not known whether there are differences in the immunity provided against these diverse invaders. We studied this issue using theStaphylococcus epidermidisCRISPR system, which harbors spacers matching both phages and plasmids. We determined that this CRISPR system provides similar levels of defense against the conjugative plasmid pG0400 and the bacteriophage CNPX. However, whereas antiplasmid immunity was very sensitive to the introduction of mismatches in the target sequence, mutations in the phage target were largely tolerated. Placing the phage and plasmid targets into a vector that can be both conjugated and transduced, we demonstrated that the route of entry of the target has no impact on the effect of the mismatches on immunity. Instead, we established that the specific sequences of each spacer/target determine the susceptibility of theS. epidermidisCRISPR system to mutations. Therefore, spacers that are more resistant to mismatches would provide long-term immunity against phages and plasmids that otherwise would escape CRISPR targeting through the accumulation of mutations in the target sequence. These results uncover an unexpected complexity in the arms race between CRISPR-Cas systems and prokaryotic infectious genetic elements.IMPORTANCECRISPR-Cas loci protect bacteria and archaea from both phage infection and plasmid invasion. These loci harbor short sequences of phage and plasmid origin known as “spacers” that specify the targets of CRISPR-Cas immunity. The presence of a spacer sequence matching a phage or plasmid ensures host immunity against infection by these genetic elements. In turn, phages and plasmids constantly mutate their targets to avoid recognition by the spacers of the CRISPR-Cas immune system. In this study, we demonstrated that different spacer sequences vary in their ability to tolerate target mutations that allow phages and plasmids to escape from CRISPR-Cas immunity. These results uncover an unexpected complexity in the arms race between CRISPR-Cas systems and prokaryotic infectious genetic elements.

scholarly journals Identification of Spacer and Protospacer Sequence Requirements in the Vibrio cholerae Type I-E CRISPR/Cas System

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Jacob Bourgeois ◽  
David W. Lazinski ◽  
Andrew Camilli
Keyword(s):  
Immune System ◽  
Vibrio Cholerae ◽  
Molecular Mechanisms ◽  
Bacterial Species ◽  
Adaptive Immune System ◽  
Initial Study ◽  
Type I ◽  
Content Type ◽  
Protospacer Adjacent Motif ◽  
Sequence Elements

ABSTRACT The prokaryotic adaptive immune system CRISPR/Cas serves as a defense against bacteriophage and invasive nucleic acids. A type I-E CRISPR/Cas system has been detected in classical biotype isolates of Vibrio cholerae, the causative agent of the disease cholera. Experimental characterization of this system revealed a functional immune system that operates using a 5′-TT-3′ protospacer-adjacent motif (PAM) for interference. However, several designed spacers against the 5′-TT-3′ PAM do not interfere as expected, indicating that further investigation of this system is necessary. In this study, we identified additional conserved sequences, including a pyrimidine in the 5′ position of the spacer and a purine in the complementary position of the protospacer using 873 unique spacers and 2,267 protospacers mined from CRISPR arrays in deposited sequences of V. cholerae. We present bioinformatic evidence that during acquisition the protospacer purine is captured in the prespacer and that a 5′-RTT-3′ PAM is necessary for spacer acquisition. Finally, we demonstrate experimentally, by designing and manipulating spacer and cognate PAMs in a plasmid conjugation assay, that a 5′-RTT-3′ PAM is necessary for CRISPR interference, and we discover functional consequences for spacer efficacy related to the identity of the 5′ spacer pyrimidine. IMPORTANCE Bacterial CRISPR/Cas systems provide immunity by defending against phage and other invading elements. A thorough comprehension of the molecular mechanisms employed by these diverse systems will improve our understanding of bacteriophage-bacterium interactions and bacterial adaptation to foreign DNA. The Vibrio cholerae type I-E system was previously identified in an extinct classical biotype and was partially characterized for its function. Here, using both bioinformatic and functional assays, we extend that initial study. We have found that the type I-E system still exists in modern strains of V. cholerae. Furthermore, we defined additional sequence elements both in the CRISPR array and in target DNA that are required for immunity. CRISPR/Cas systems are now commonly used as precise and powerful genetic engineering tools. Knowledge of the sequences required for CRISPR/Cas immunity is a prerequisite for the effective design and experimental use of these systems. Our results greatly facilitate the effective use of one such system. Furthermore, we provide a publicly available software program that assists in the detection and validation of CRISPR/Cas immunity requirements when such a system exists in a bacterial species.

scholarly journals Structure and Evolution of Chlorate Reduction Composite Transposons

mBio ◽  
2013 ◽  
Vol 4 (4) ◽  
Author(s):  
Iain C. Clark ◽  
Ryan A. Melnyk ◽  
Anna Engelbrektson ◽  
John D. Coates
Keyword(s):  
Phylogenetic Analysis ◽  
Comparative Analysis ◽  
Dimethyl Sulfoxide ◽  
De Novo ◽  
Mobile Element ◽  
Insertion Sequences ◽  
Type Ii ◽  
Bacterial Strains ◽  
Chlorite Dismutase ◽  
Content Type

ABSTRACTThe genes for chlorate reduction in six bacterial strains were analyzed in order to gain insight into the metabolism. A newly isolated chlorate-reducing bacterium (Shewanella algaeACDC) and three previously isolated strains (Ideonella dechloratans,Pseudomonassp. strain PK, andDechloromarinus chlorophilusNSS) were genome sequenced and compared to published sequences (Alicycliphilus denitrificansBC plasmid pALIDE01 andPseudomonas chloritidismutansAW-1).De novoassembly of genomes failed to join regions adjacent to genes involved in chlorate reduction, suggesting the presence of repeat regions. Using a bioinformatics approach and finishing PCRs to connect fragmented contigs, we discovered that chlorate reduction genes are flanked by insertion sequences, forming composite transposons in all four newly sequenced strains. These insertion sequences delineate regions with the potential to move horizontally and define a set of genes that may be important for chlorate reduction. In addition to core metabolic components, we have highlighted several such genes through comparative analysis and visualization. Phylogenetic analysis places chlorate reductase within a functionally diverse clade of type II dimethyl sulfoxide (DMSO) reductases, part of a larger family of enzymes with reactivity toward chlorate. Nucleotide-level forensics of regions surrounding chlorite dismutase (cld), as well as its phylogenetic clustering in a betaproteobacterial Cld clade, indicate thatcldhas been mobilized at least once from a perchlorate reducer to build chlorate respiration.IMPORTANCEGenome sequencing has identified, for the first time, chlorate reduction composite transposons. These transposons are constructed with flanking insertion sequences that differ in type and orientation between organisms, indicating that this mobile element has formed multiple times and is important for dissemination. Apart from core metabolic enzymes, very little is known about the genetic factors involved in chlorate reduction. Comparative analysis has identified several genes that may also be important, but the relative absence of accessory genes suggests that this mobile metabolism relies on host systems for electron transport, regulation, and cofactor synthesis. Phylogenetic analysis of Cld and ClrA provides support for the hypothesis that chlorate reduction was built multiple times from type II dimethyl sulfoxide (DMSO) reductases andcld. In at least one case,cldhas been coopted from a perchlorate reduction island for this purpose. This work is a significant step toward understanding the genetics and evolution of chlorate reduction.

scholarly journals Characterization of a Type II-A CRISPR-Cas System in Streptococcus mutans

mSphere ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Cas Mosterd ◽  
Sylvain Moineau
Keyword(s):  
Streptococcus Mutans ◽  
Defense Mechanisms ◽  
Bacterial Species ◽  
Oral Microbiota ◽  
Type Ii ◽  
Content Type ◽  
C Terminus ◽  
Protospacer Adjacent Motif ◽  
Cas9 Protein

ABSTRACT Streptococcus mutans and its virulent phages are important members of the human oral microbiota. S. mutans is also the primary causal agent of dental caries. To survive in this ecological niche, S. mutans must encode phage defense mechanisms, which include CRISPR-Cas systems. Here, we describe the CRISPR-Cas type II-A system of S. mutans strain P42S, which was found to display natural adaptation and interference activity in response to phage infection and plasmid transformation. Newly acquired spacers were integrated both at the 5′ end of the CRISPR locus and ectopically. In comparisons of the cas genes of P42S to those of other strains of S. mutans, cas1, cas2, and csn2 appear to be highly conserved within the species. However, more diversity was observed with cas9. While the nuclease domains of S. mutans Cas9 (SmCas9) are conserved, the C terminus of the protein, including the protospacer adjacent motif (PAM) recognition domain, is less conserved. In support of these findings, we experimentally demonstrated that the PAMs associated with SmCas9 of strain P42S are NAA and NGAA. These PAMs are different from those previously reported for the CRISPR-Cas system of the model strain S. mutans UA159. This study illustrates the diversity of CRISPR-Cas type II-A systems that can be found within the same bacterial species. IMPORTANCE CRISPR-Cas is one of the mechanisms used by bacteria to defend against viral predation. Increasing our knowledge of the biology and diversity of CRISPR-Cas systems will also improve our understanding of virus-bacterium interactions. As CRISPR-Cas systems acquiring novel immunities under laboratory conditions are rare, Streptococcus mutans strain P42S provides an alternative model to study the adaptation step, which is still the least understood step in CRISPR-Cas biology. Furthermore, the availability of a natural Cas9 protein recognizing an AT-rich PAM opens up new avenues for genome editing purposes.

scholarly journals Cas4 Nucleases Can Effect Specific Integration of CRISPR Spacers

2019 ◽  
Vol 201 (12) ◽  
Author(s):  
Zhufeng Zhang ◽  
Saifu Pan ◽  
Tao Liu ◽  
Yingjun Li ◽  
Nan Peng
Keyword(s):  
Amino Acid ◽  
Amino Acid Residues ◽  
Spacer Length ◽  
Content Type ◽  
Sulfolobus Islandicus ◽  
Regulator Protein ◽  
Identify Amino Acid ◽  
Protospacer Adjacent Motif ◽  
Short Palindromic Repeat

ABSTRACT Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems incorporate short DNA fragments from invasive genetic elements into host CRISPR arrays in order to generate host immunity. Recently, we demonstrated that the Csa3a regulator protein triggers CCN protospacer-adjacent motif (PAM)-dependent CRISPR spacer acquisition in the subtype I-A CRISPR-Cas system of Sulfolobus islandicus. However, the mechanisms underlying specific protospacer selection and spacer insertion remained unclear. Here, we demonstrate that two Cas4 family proteins (Cas4 and Csa1) have essential roles (i) in recognizing the 5′ PAM and 3′ nucleotide motif of protospacers and (ii) in determining both the spacer length and its orientation. Furthermore, we identify amino acid residues of the Cas4 proteins that facilitate these functions. Overexpression of the Cas4 and Csa1 proteins, and also that of an archaeal virus-encoded Cas4 protein, resulted in strongly reduced adaptation efficiency, and the former proteins yielded a high incidence of PAM-dependent atypical spacer integration or of PAM-independent spacer integration. We further demonstrated that in plasmid challenge experiments, overexpressed Cas4-mediated defective spacer acquisition in turn potentially enabled targeted DNA to escape subtype I-A CRISPR-Cas interference. In summary, these results define the specific involvement of diverse Cas4 proteins in in vivo CRISPR spacer acquisition. Furthermore, we provide support for an anti-CRISPR role for virus-encoded Cas4 proteins that involves compromising CRISPR-Cas interference activity by hindering spacer acquisition. IMPORTANCE The Cas4 family endonuclease is an essential component of the adaptation module in many variants of CRISPR-Cas adaptive immunity systems. The Crenarchaeota Sulfolobus islandicus REY15A carries two cas4 genes (cas4 and csa1) linked to the CRISPR arrays. Here, we demonstrate that Cas4 and Csa1 are essential to CRISPR spacer acquisition in this organism. Both proteins specify the upstream and downstream conserved nucleotide motifs of the protospacers and define the spacer length and orientation in the acquisition process. Conserved amino acid residues, in addition to those recently reported, were identified to be important for these functions. More importantly, overexpression of the Sulfolobus viral Cas4 abolished spacer acquisition, providing support for an anti-CRISPR role for virus-encoded Cas4 proteins that inhibit spacer acquisition.

scholarly journals Expression of Phosphocitrate-Targeted Genes in Osteoarthritis Menisci

2014 ◽  
Vol 2014 ◽  
pp. 1-17 ◽  
Author(s):  
Yubo Sun ◽  
David R. Mauerhan ◽  
Nury M. Steuerwald ◽  
Jane Ingram ◽  
Jeffrey S. Kneisl ◽  
...  
Keyword(s):  
Immune System ◽  
Molecular Mechanisms ◽  
Fibroblast Growth Factor Receptor ◽  
Collagen Type ◽  
Skeletal Development ◽  
Growth Factor Receptor ◽  
Type Ii ◽  
Immune System Activation ◽  
System Activation ◽  
Disease Modifying

Phosphocitrate (PC) inhibited calcium crystal-associated osteoarthritis (OA) in Hartley guinea pigs. However, the molecular mechanisms remain elusive. This study sought to determine PC targeted genes and the expression of select PC targeted genes in OA menisci to test hypothesis that PC exerts its disease modifying activity in part by reversing abnormal expressions of genes involved in OA. We found that PC downregulated the expression of numerous genes classified in immune response, inflammatory response, and angiogenesis, including chemokine (C-C motif) ligand 5, Fc fragment of IgG, low affinity IIIb receptor (FCGR3B), and leukocyte immunoglobulin-like receptor, subfamily B member 3 (LILRB3). In contrast, PC upregulated the expression of many genes classified in skeletal development, including collagen type II alpha1, fibroblast growth factor receptor 3 (FGFR3), and SRY- (sex determining region Y-) box 9 (SOX-9). Immunohistochemical examinations revealed higher levels of FCGR3B and LILRB3 and lower level of SOX-9 in OA menisci. These findings indicate that OA is a disease associated with immune system activation and decreased expression of SOX-9 gene in OA menisci. PC exerts its disease modifying activity on OA, at least in part, by targeting immune system activation and the production of extracellular matrix and selecting chondroprotective proteins.

Split spinal cord malformations in children

1998 ◽  
Vol 88 (1) ◽  
pp. 57-65 ◽  
Author(s):  
Yusuf Ersşahin ◽  
Saffet Mutluer ◽  
Sevgül Kocaman ◽  
Eren Demirtasş
Keyword(s):  
Spinal Cord ◽  
Neurological Deficits ◽  
Single Type ◽  
Type I ◽  
Type Ii ◽  
Content Type ◽  
Spinal Lesion ◽  
The Mean ◽  
Fine Print

Object. The authors reviewed and analyzed information on 74 patients with split spinal cord malformations (SSCMs) treated between January 1, 1980 and December 31, 1996 at their institution with the aim of defining and classifying the malformations according to the method of Pang, et al. Methods. Computerized tomography myelography was superior to other radiological tools in defining the type of SSCM. There were 46 girls (62%) and 28 boys (38%) ranging in age from less than 1 day to 12 years (mean 33.08 months). The mean age (43.2 months) of the patients who exhibited neurological deficits and orthopedic deformities was significantly older than those (8.2 months) without deficits (p = 0.003). Fifty-two patients had a single Type I and 18 patients a single Type II SSCM; four patients had composite SSCMs. Sixty-two patients had at least one associated spinal lesion that could lead to spinal cord tethering. After surgery, the majority of the patients remained stable and clinical improvement was observed in 18 patients. Conclusions. The classification of SSCMs proposed by Pang, et al., will eliminate the current chaos in terminology. In all SSCMs, either a rigid or a fibrous septum was found to transfix the spinal cord. There was at least one unrelated lesion that caused tethering of the spinal cord in 85% of the patients. The risk of neurological deficits resulting from SSCMs increases with the age of the patient; therefore, all patients should be surgically treated when diagnosed, especially before the development of orthopedic and neurological manifestations.

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