海南海口温泉真菌、细菌多样性及其环境影响因素分析

【目的】海南海口含有丰富的温泉资源,对温泉微生物多样性进行研究,有助于进一步开发和利用海南温泉微生物资源。【方法】本文采用Illumina Hi Seq高通量测序技术对海口3个温泉[海甸岛荣域温泉(S1)、火山口开心农场温泉(S2)和西海岸海长流温泉(S3)]水样中微生物ITS序列和16Sr RNA基因V3-V4区进行测序及生物信息学分析,探究海口市3个不同区域的温泉真菌多样性与细菌多样性。【结果】(1)α多样性分析表明,真菌群落中,S3(29)S1(29)S2,而在细菌群落中,S2(29)S1(29)S3。β多样性分析表明,3个温泉真菌群落和细菌群落组成差异皆显著。(2)分类分析表明,温泉真菌群落优势菌门为子囊菌门(Ascomycota)和担子菌门(Basidiomycota),细菌群落优势菌门为变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、Thermi、硝化螺旋菌门(Nitrospirae)、绿菌门(Chlorobi)、厚壁菌门(Firmicutes)、绿弯菌门(Chloroflexi)、放线菌门(Actinobacteria)。(3) CCA (Canonical correspondence analysis)分析表明,3个温泉的真菌群落主要影响因子是温度,细菌群落主要影响因子是总磷。【结论】海南省海口市温泉中含有丰富的微生物资源,其微生物群落组成受多种环境因子影响,且影响真菌和细菌的主要环境因子不同。     

Characterization of CRISPR-Cas systems in the Ralstonia solanacearum species complex

Clustered regularly interspaced short palindromic repeats (CRISPRs) are composed of an array of short DNA repeat sequences separated by unique spacer sequences that are flanked by associated (Cas) genes. CRISPR-Cas systems are found in the genomes of several microbes and can act as an adaptive immune mechanism against invading foreign nucleic acids, such as phage genomes. Here, we studied the CRISPR-Cas systems in plant-pathogenic bacteria of the Ralstonia solanacearum species complex (RSSC). A CRISPR-Cas system was found in 31% of RSSC genomes present in public databases. Specifically, CRISPR-Cas types I-E and II-C were found, with I-E being the most common. The presence of the same CRISPR-Cas types in distinct Ralstonia phylotypes and species suggests the acquisition of the system by a common ancestor before Ralstonia species segregation. In addition, a Cas1 phylogeny (I-E type) showed a perfect geographical segregation of phylotypes, supporting an ancient acquisition. Ralstoniasolanacearum strains CFBP2957 and K60^T were challenged with a virulent phage, and the CRISPR arrays of bacteriophage-insensitive mutants (BIMs) were analysed. No new spacer acquisition was detected in the analysed BIMs. The functionality of the CRISPR-Cas interference step was also tested in R. solanacearum CFBP2957 using a spacer-protospacer adjacent motif (PAM) delivery system, and no resistance was observed against phage phiAP1. Our results show that the CRISPR-Cas system in R. solanacearum CFBP2957 is not its primary antiviral strategy.     

CRISPR Content Correlates with the Pathogenic Potential of Escherichia coli

Guide RNA molecules (crRNA) produced from clustered regularly interspaced short palindromic repeat (CRISPR) arrays, altogether with effector proteins (Cas) encoded by cognate cas (CRISPR associated) genes, mount an interference mechanism (CRISPR-Cas) that limits acquisition of foreign DNA in Bacteria and Archaea. The specificity of this action is provided by the repeat intervening spacer carried in the crRNA, which upon hybridization with complementary sequences enables their degradation by a Cas endonuclease. Moreover, CRISPR arrays are dynamic landscapes that may gain new spacers from infecting elements or lose them for example during genome replication. Thus, the spacer content of a strain determines the diversity of sequences that can be targeted by the corresponding CRISPR-Cas system reflecting its functionality. Most Escherichia coli strains possess either type I-E or I-F CRISPR-Cas systems. To evaluate their impact on the pathogenicity of the species, we inferred the pathotype and pathogenic potential of 126 strains of this and other closely related species and analyzed their repeat content. Our results revealed a negative correlation between the number of I-E CRISPR units in this system and the presence of pathogenicity traits: the median number of repeats was 2.5-fold higher for commensal isolates (with 29.5 units, range 0–53) than for pathogenic ones (12.0, range 0–42). Moreover, the higher the number of virulence factors within a strain, the lower the repeat content. Additionally, pathogenic strains of distinct ecological niches (i.e., intestinal or extraintestinal) differ in repeat counts. Altogether, these findings support an evolutionary connection between CRISPR and pathogenicity in E. coli.     

Active site plasticity enables metal-dependent tuning of Cas5d nuclease activity in CRISPR-Cas type I-C system

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) in association with CRISPR-associated (Cas) proteins constitutes a formidable defense system against mobile genetic elements in prokaryotes. In type I-C, the ribonucleoprotein surveillance complex comprises only three Cas proteins, namely, Cas5d, Csd1 and Csd2. Unlike type I-E that uses Cse3/CasE for metal-independent CRISPR RNA maturation, type I-C that lacks this deputes Cas5d to process the pre-crRNA. Here, we report the promiscuous DNase activity of Cas5d in presence of divalent metals. Remarkably, the active site that renders RNA hydrolysis may be tuned by metal to act on DNA substrates too. Further, the realization that Csd1 is a fusion of its functional homolog Cse1/CasA and Cse2/CasB forecasts that the stoichiometry of the constituents of the surveillance complex in type I-C may differ from type I-E. Although Csd2 seems to be inert, Csd1 too exhibits RNase and metal-dependent DNase activity. Thus, in addition to their proposed functions, the DNase activity of Cas5d and Csd1 may also enable them to be co-opted in adaptation and interference stages of CRISPR immunity wherein interaction with DNA substrates is involved.     

Cas5d Protein Processes Pre-crRNA and Assembles into a Cascade-like Interference Complex in Subtype I-C/Dvulg CRISPR-Cas System

Clustered regularly interspaced short palindromic repeats (CRISPRs), together with an operon of CRISPR-associated (Cas) proteins, form an RNA-based prokaryotic immune system against exogenous genetic elements. Cas5 family proteins are found in several type I CRISPR-Cas systems. Here, we report the molecular function of subtype I-C/Dvulg Cas5d from Bacillus halodurans. We show that Cas5d cleaves pre-crRNA into unit length by recognizing both the hairpin structure and the 3' single stranded sequence in the CRISPR repeat region. Cas5d structure reveals a ferredoxin domain-based architecture and a catalytic triad formed by Y46, K116, and H117 residues. We further show that after pre-crRNA processing, Cas5d assembles with crRNA, Csd1, and Csd2 proteins to form a multi-sub-unit interference complex similar to Escherichia coli Cascade (CRISPR-associated complex for antiviral defense) in architecture. Our results suggest that formation of a crRNA-presenting Cascade-like complex is likely a common theme among type I CRISPR subtypes.     

CRISPR-Cas系统转录调控机制的研究进展

原核生物可利用由CRISPR-Cas系统(clustered regularly interspaced short palindromic repeats-CRISPR associated)介导的适应性免疫机制防御外源核酸入侵.在适应性免疫过程中,原核生物将外源核酸部分片段整合至自身CRISPR阵列中,表达并加工的...     

Comparative genomic structures of Mycobacterium CRISPR-Cas

Clustered regularly interspaced short palindromic repeats (CRISPR) are inheritable genetic elements of many archaea and bacteria, conferring acquired immunity against invading nucleic acids. CRISPR might be indicative of the bacterial niche adaptation and evolutionary. Mycobacterium is an important genus occupying diverse niches with profound medical and environmental significance. To present a comparative genomic landscape of the Mycobacterium CRISPR, the feature of mycobacterium CRISPR structures with sequenced complete genomes were bioinformatically analyzed. The results show that CRISPR structures can be found among 14 mycobacteria, and all loci are chromosomally located. Long CRISPRs present in three species, namely M. tuberculosis, M. bovis, and M. avium. Integrated CRISPR-Cas system can only be found in M. tuberculosis and M. bovis, with highly conserved repeat sequences, very short leaders, and promoterless. M. tuberculosis and M. bovis repeat sequences cannot form stable RNA secondary structure, consistent with a Cas6-binding sequence. M. avium repeat sequences can form classical stem-loop structure. A three-step model of M. tuberculosis CRISPR-Cas system action was put forward based on the composition and function of cas genes cluster. M. tuberculosis and M. bovis CRISPRs might interfere with the invading nucleic acids, but have somehow lost the capacity to incorporate new spacers and co-evolve with corresponding mycobacteriophages.     

Targeted large fragment deletion in plants using paired crRNAs with type I CRISPR system

The CRISPR-Cas systems have been widely used as genome editing tools, with type II and V systems typically introducing small indels, and type I system mediating long-range deletions. However, the precision of type I systems for large fragment deletion is still remained to be optimized. Here, we developed a compact Cascade-Cas3 Dvu I-C system with Cas11c for plant genome editing. The Dvu I-C system was efficient to introduce controllable large fragment deletion up to at least 20 kb using paired crRNAs. The paired-crRNAs design also improved the controllability of deletions for the type I-E system. Dvu I-C system was sensitive to spacer length and mismatch, which was benefit for target specificity. In addition, we showed that the Dvu I-C system was efficient for generating stable transgenic lines in maize and rice with the editing efficiency up to 86.67%. Overall, Dvu I-C system we developed here is powerful for achieving controllable large fragment deletions.     

The structure of AcrIE4-F7 reveals a common strategy for dual CRISPR inhibition by targeting PAM recognition sites

Bacteria and archaea use the CRISPR-Cas system to fend off invasions of bacteriophages and foreign plasmids. In response, bacteriophages encode anti-CRISPR (Acr) proteins that potently inhibit host Cas proteins to suppress CRISPR-mediated immunity. AcrIE4-F7, which was isolated from Pseudomonas citronellolis, is a fused form of AcrIE4 and AcrIF7 that inhibits both type I-E and type I-F CRISPR-Cas systems. Here, we determined the structure of AcrIE4-F7 and identified its Cas target proteins. The N-terminal AcrIE4 domain adopts a novel α-helical fold that targets the PAM interaction site of the type I-E Cas8e subunit. The C-terminal AcrIF7 domain exhibits an αβ fold like native AcrIF7, which disables target DNA recognition by the PAM interaction site in the type I-F Cas8f subunit. The two Acr domains are connected by a flexible linker that allows prompt docking onto their cognate Cas8 targets. Conserved negative charges in each Acr domain are required for interaction with their Cas8 targets. Our results illustrate a common mechanism by which AcrIE4-F7 inhibits divergent CRISPR-Cas types.     

A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes

Clustered regularly interspaced short palindromic repeats (CRISPRs) are a family of DNA direct repeats found in many prokaryotic genomes. Repeats of 21-37 bp typically show weak dyad symmetry and are separated by regularly sized, nonrepetitive spacer sequences. Four CRISPR-associated (Cas) protein families, designated Cas1 to Cas4, are strictly associated with CRISPR elements and always occur near a repeat cluster. Some spacers originate from mobile genetic elements and are thought to confer "immunity" against the elements that harbor these sequences. In the present study, we have systematically investigated uncharacterized proteins encoded in the vicinity of these CRISPRs and found many additional protein families that are strictly associated with CRISPR loci across multiple prokaryotic species. Multiple sequence alignments and hidden Markov models have been built for 45 Cas protein families. These models identify family members with high sensitivity and selectivity and classify key regulators of development, DevR and DevS, in Myxococcus xanthus as Cas proteins. These identifications show that CRISPR/cas gene regions can be quite large, with up to 20 different, tandem-arranged cas genes next to a repeat cluster or filling the region between two repeat clusters. Distinctive subsets of the collection of Cas proteins recur in phylogenetically distant species and correlate with characteristic repeat periodicity. The analyses presented here support initial proposals of mobility of these units, along with the likelihood that loci of different subtypes interact with one another as well as with host cell defensive, replicative, and regulatory systems. It is evident from this analysis that CRISPR/cas loci are larger, more complex, and more heterogeneous than previously appreciated.     

CRISPR-Cas systems in the marine actinomycete Salinispora: linkages with phage defense,microdiversity and biogeography

Background

Prokaryotic CRISPR-Cas systems confer resistance to viral infection and thus mediate bacteria-phage interactions. However, the distribution and functional diversity of CRISPRs among environmental bacteria remains largely unknown. Here, comparative genomics of 75 Salinispora strains provided insight into the diversity and distribution of CRISPR-Cas systems in a cosmopolitan marine actinomycete genus.

Results

CRISPRs were found in all Salinispora strains, with the majority containing multiple loci and different Cas array subtypes. Of the six subtypes identified, three have not been previously described. A lower prophage frequency in S. arenicola was associated with a higher fraction of spacers matching Salinispora prophages compared to S. tropica, suggesting differing defensive capacities between Salinispora species. The occurrence of related prophages in strains from distant locations, as well as spacers matching those prophages inserted throughout spacer arrays, indicate recurring encounters with widely distributed phages over time. Linkages of CRISPR features with Salinispora microdiversity pointed to subclade-specific contacts with mobile genetic elements (MGEs). This included lineage-specific spacer deletions or insertions, which may reflect weak selective pressures to maintain immunity or distinct temporal interactions with MGEs, respectively. Biogeographic patterns in spacer and prophage distributions support the concept that Salinispora spp. encounter localized MGEs. Moreover, the presence of spacers matching housekeeping genes suggests that CRISPRs may have functions outside of viral defense.

Conclusions

This study provides a comprehensive examination of CRISPR-Cas systems in a broadly distributed group of environmental bacteria. The ubiquity and diversity of CRISPRs in Salinispora suggests that CRISPR-mediated interactions with MGEs represent a major force in the ecology and evolution of this cosmopolitan marine actinomycete genus.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-936) contains supplementary material, which is available to authorized users.     

On the global CRISPR array behavior in class I systems

Background

Much effort is underway to build and upgrade databases and tools related to occurrence, diversity, and characterization of CRISPR-Cas systems. As microbial communities and their genome complements are unearthed, much emphasis has been placed on details of individual strains and model systems within the CRISPR-Cas classification, and that collection of information as a whole affords the opportunity to analyze CRISPR-Cas systems from a quantitative perspective to gain insight into distribution of CRISPR array sizes across the different classes, types and subtypes. CRISPR diversity, nomenclature, occurrence, and biological functions have generated a plethora of data that created a need to understand the size and distribution of these various systems to appreciate their features and complexity.

Results

By utilizing a statistical framework and visual analytic techniques, we have been able to test several hypotheses about CRISPR loci in bacterial class I systems. Quantitatively, though CRISPR loci can expand to hundreds of spacers, the mean and median sizes are 40 and 25, respectively, reflecting rather modest acquisition and/or retention overall. Histograms uncovered that CRISPR array size displayed a parametric distribution, which was confirmed by a goodness-of fit test. Mapping the frequency of CRISPR loci on a standardized chromosome plot revealed that CRISPRs have a higher probability of occurring at clustered locations along the positive or negative strand. Lastly, when multiple arrays occur in a particular system, the size of a particular CRISPR array varies with its distance from the cas operon, reflecting acquisition and expansion biases.

Conclusions

This study establishes that bacterial Class I CRISPR array size tends to follow a geometric distribution; these CRISPRs are not randomly distributed along the chromosome; and the CRISPR array closest to the cas genes is typically larger than loci in trans. Overall, we provide an analytical framework to understand the features and behavior of CRISPR-Cas systems through a quantitative lens.

Reviewers

This article was reviewed by Eugene Koonin (NIH-NCBI) and Uri Gophna (Tel Aviv University).

     

Advances in CRISPR-Cas systems for RNA targeting,tracking and editing

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, especially type II (Cas9) systems, have been widely used in gene/genome targeting. Modifications of Cas9 enable these systems to become platforms for precise DNA manipulations. However, the utilization of CRISPR-Cas systems in RNA targeting remains preliminary. The discovery of type VI CRISPR-Cas systems (Cas13) shed light on RNA-guided RNA targeting. Cas13d, the smallest Cas13 protein, with a length of only ~930 amino acids, is a promising platform for RNA targeting compatible with viral delivery systems. Much effort has also been made to develop Cas9, Cas13a and Cas13b applications for RNA-guided RNA targeting. The discovery of new RNA-targeting CRISPR-Cas systems as well as the development of RNA-targeting platforms with Cas9 and Cas13 will promote RNA-targeting technology substantially. Here, we review new advances in RNA-targeting CRISPR-Cas systems as well as advances in applications of these systems in RNA targeting, tracking and editing. We also compare these Cas protein-based technologies with traditional technologies for RNA targeting, tracking and editing. Finally, we discuss remaining questions and prospects for the future.     

Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition,bi-directionally from the primed protospacer

Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼350 new spacers acquired in priming events and identified a 5′-protospacer-GG-3′ protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2–3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.     

Diverse CRISPRs evolving in human microbiomes

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, together with cas (CRISPR-associated) genes, form the CRISPR/Cas adaptive immune system, a primary defense strategy that eubacteria and archaea mobilize against foreign nucleic acids, including phages and conjugative plasmids. Short spacer sequences separated by the repeats are derived from foreign DNA and direct interference to future infections. The availability of hundreds of shotgun metagenomic datasets from the Human Microbiome Project (HMP) enables us to explore the distribution and diversity of known CRISPRs in human-associated microbial communities and to discover new CRISPRs. We propose a targeted assembly strategy to reconstruct CRISPR arrays, which whole-metagenome assemblies fail to identify. For each known CRISPR type (identified from reference genomes), we use its direct repeat consensus sequence to recruit reads from each HMP dataset and then assemble the recruited reads into CRISPR loci; the unique spacer sequences can then be extracted for analysis. We also identified novel CRISPRs or new CRISPR variants in contigs from whole-metagenome assemblies and used targeted assembly to more comprehensively identify these CRISPRs across samples. We observed that the distributions of CRISPRs (including 64 known and 86 novel ones) are largely body-site specific. We provide detailed analysis of several CRISPR loci, including novel CRISPRs. For example, known streptococcal CRISPRs were identified in most oral microbiomes, totaling ～8,000 unique spacers: samples resampled from the same individual and oral site shared the most spacers; different oral sites from the same individual shared significantly fewer, while different individuals had almost no common spacers, indicating the impact of subtle niche differences on the evolution of CRISPR defenses. We further demonstrate potential applications of CRISPRs to the tracing of rare species and the virus exposure of individuals. This work indicates the importance of effective identification and characterization of CRISPR loci to the study of the dynamic ecology of microbiomes.