Direct Repeat Sequences Research Articles

Fine-tuning the evolutionary stability of recombinant herpesviral transmissible vaccines.

Spillover of infectious diseases from wild animal populations constitutes a long-standing threat to human health for which few globally viable solutions have been developed. The use of oral baits laden with conventional vaccines distributed en masse represents one success story but is costly and practicable primarily for rabies risk reduction in North American and European carnivores. Efforts to expand vaccination to control pathogens within less accessible wildlife populations have raised interest in a new kind of vaccine capable of spreading pathogen-specific immunity through autonomous spread. However, such 'transmissible' vaccines raise concerns about the irrevocable release of genetically modified viruses into the environment. Herein, we explore the feasibility of an intrinsic strategy for transgene control within these vaccines based on the genetic destabilizing effect of cis-acting sequences flanking the heterologous transgene of interest. While suitable for the control of transgene stability within all types of DNA-viral vectored vaccines, this strategy has particular applicability to transmissible vaccines. Using a combination of experiments, mathematical modelling and whole-genome sequencing, we show that the rate of transgene loss can be controlled by varying the lengths of the direct repeat sequences. This opens a way for fine-tuning the lifespan of a transmissible vaccine in the wild.

CRISPR-Cas systems in enterococci.

Enterococci are members of the microbiota of humans and other animals. They can also be found in the environment, associated with food, healthcare infections, and hospital settings. Due to their wide distribution, they are inserted in the One Health context. The selective pressure caused by the extensive use of antimicrobial agents in humans, animals, and agriculture has increased the frequency of resistance to various drugs among enterococcal species. CRISPR-Cas system, an important prokaryotic defense mechanism against the entry of mobile genetic elements, may prevent the acquisition of genes involved in antimicrobial resistance and virulence. This system has been increasingly used as a gene editing tool, which can be used as a way to recognize and inactivate genes of interest. Here, we conduct a review on CRISPR systems found in enterococci, considering their occurrence, structure and organization, mechanisms of action and use as a genetic engineering technology. Type II-A CRISPR-Cas systems were shown to be the most frequent among enterococcal species, and the orphan CRISPR2 was the most commonly found system (54.1%) among enterococcal species, especially in Enterococcus faecalis. Distribution of CRISPR systems varied among species. CRISPR systems had 1 to 20 spacers, with size between 23 and 37bp and direct repeat sequences from 25 to 37bp. Several applications of the CRISPR-Cas biotechnology have been described in enterococci, mostly in vitro, using this editing tool to target resistance- and virulence-related genes.

CRISETR: an efficient technology for multiplexed refactoring of biosynthetic gene clusters.

The efficient refactoring of natural product biosynthetic gene clusters (BGCs) for activating silent BGCs is a central challenge for the discovery of new bioactive natural products. Herein, we have developed a simple and robust CRISETR (CRISPR/Cas9 and RecET-mediated Refactoring) technique, combining clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 and RecET, for the multiplexed refactoring of natural product BGCs. By this approach, natural product BGCs can be refactored through the synergistic interaction between RecET-mediated efficient homologous recombination and the CRISPR/Cas9 system. We firstperformed a proof-of-concept validation of the ability of CRISETR, and CRISETR can achieve simultaneous replacement of four promoter sites and marker-free replacement of single promoter site in natural product BGCs. Subsequently, we applied CRISETR to the promoter engineering of the 74-kb daptomycin BGC containing a large number of direct repeat sequences forenhancingthe heterologous production of daptomycin. We used combinatorial design to build multiple refactored daptomycin BGCs with diverse combinations of promoters different in transcriptional strengths, and the yield of daptomycin was improved 20.4-fold in heterologous host Streptomyces coelicolor A3(2). In general, CRISETR exhibits enhanced tolerance to repetitive sequences within gene clusters, enabling efficient refactoring of diverse and complex BGCs, which would greatly accelerate discovery of novel bioactive metabolites present in microorganism.

Intra-Host Citrus Tristeza Virus Populations during Prolonged Infection Initiated by a Well-Defined Sequence Variant in Nicotiana benthamiana.

Due to the error-prone nature of viral RNA-dependent RNA polymerases, the replication of RNA viruses results in a diversity of viral genomes harboring point mutations, deletions, insertions, and genome rearrangements. Citrus tristeza virus (CTV), a causal agent of diseases of economically important citrus species, shows intrinsic genetic stability. While the virus appears to have some mechanism that limits the accumulation of single-nucleotide variants, the production of defective viral genomes (DVGs) during virus infection has been reported for certain variants of CTV. The intra-host diversity generated during plant infection with variant T36 (CTV-T36) remains unclear. To address this, we analyzed the RNA species accumulated in the initially infected and systemic leaves of Nicotiana benthamiana plants inoculated with an infectious cDNA clone of CTV-T36, which warranted that infection was initiated by a known, well-defined sequence variant of the virus. CTV-T36 limited the accumulation of single-nucleotide mutants during infection. With that, four types of DVGs-deletions, insertions, and copy- and snap-backs-were found in all the samples, with deletions and insertions being the most common types. Hot-spots across the genome for DVG recombination and short direct sequence repeats suggest that sequence complementarity could mediate DVG formation. In conclusion, our study illustrates the formation of diverse DVGs during CTV-T36 infection. To the best of our knowledge, this is the first study that has analyzed the genetic variability and recombination of a well-defined sequence variant of CTV in an herbaceous host.

Roles of Nuclear Orphan Receptors TR2 and TR4 during Hematopoiesis.

TR2 and TR4 (NR2C1 and NR2C2, respectively) are evolutionarily conserved nuclear orphan receptors capable of binding direct repeat sequences in a stage-specific manner. Like other nuclear receptors, TR2 and TR4 possess important roles in transcriptional activation or repression with developmental stage and tissue specificity. TR2 and TR4 bind DNA and possess the ability to complex with available cofactors mediating developmental stage-specific actions in primitive and definitive erythrocytes. In erythropoiesis, TR2 and TR4 are required for erythroid development, maturation, and key erythroid transcription factor regulation. TR2 and TR4 recruit and interact with transcriptional corepressors or coactivators to elicit developmental stage-specific gene regulation during hematopoiesis.

Bacillus phage phi18-2 is a novel temperate virus with an unintegrated genome present in the cytoplasm of lysogenic cells as a linear phage-plasmid.

Bacillus subtilis is a Gram-positive bacterium that is widely used in fermentation and in the pharmaceutical industry. Phage contamination occasionally occurs in various fermentation processes and causes significant economic loss. Here, we report the isolation and characterization of a temperate B. subtilis phage, termed phi18-2, from spore powder manufactured in a fermentation plant. Transmission electron microscopy showed that phi18-2 has a symmetrical polyhedral head and a long noncontractile tail. Receptor analysis showed that phi18-2 recognizes wall teichoic acid (WTA) for infection. The phage virions have a linear double-stranded DNA genome of 64,467 bp with identical direct repeat sequences of 309 bp at each end of the genome. In lysogenic cells, the phage genome was found to be present in the cytoplasm without integration into the host cell chromosome, and possibly as a linear phage-plasmid with unmodified ends. Our data may provide some insight into the molecular basis of the unique lysogenic cycle of phage phi18-2.

Characteristics of the a sequence of the duck Plague virus genome and specific cleavage of the viral genome based on the a sequence

During the replication process, the herpesvirus genome forms the head-to-tail linked concatemeric genome, which is then cleaved and packaged into the capsid. The cleavage and packing process is carried out by the terminase complex, which specifically recognizes and cleaves the concatemeric genome. This process is governed by a cis-acting sequence in the genome, named the a sequence. The a sequence and genome cleavage have been described in some herpesviruses, but it remains unclear in duck plague virus. In this study, we analysed the location, composition, and conservation of a sequence in the duck plague virus genome. The structure of the DPV genome has an a sequence of (DR4)m-(DR2)n-pac1-S termini (32 bp)-L termini (32 bp)-pac2, and the length is 841 bp. Direct repeat (DR) sequences are conserved in different DPV strains, but the number of DR copies is inconsistent. Additionally, the typical DR1 sequence was not found in the DPV a sequence. The Pac1 and pac2 motifs are relatively conserved between DPV and other herpesviruses. Cleavage of the DPV concatemeric genome was detected, and the results showed that the DPV genome can form a concatemer and is cleaved into a monomer at a specific site. We also established a sensitive method, TaqMan dual qRT‒PCR, to analyse genome cleavage. The ratio of concatemer to total viral genome was decreased during the replication process. These results will be critical for understanding the process of DPV genome cleavage, and the application of TaqMan dual qRT‒PCR will greatly facilitate more in-depth research.

Psychrophilic phage phiGM22-3 efficiently controls Pseudomonas fluorescens contamination in cold-stored milk

Pseudomonas fluorescens is a common spoilage causing microbe found in milk. Antibiotic preservatives may cause emergence of multidrug resistance, posing food safety related risks to public health. Phage treatment may be used as an alternative to antibiotics in controlling P. fluorescens contaminations. Here we reported that P. fluorescens phage phiGM22-3 reproduced rapidly over a broad temperature range of 4 through 30°C, and the optimum growth of phiGM22-3 occurred at 10°C, indicating that it was a psychrophilic virus. Genome analysis revealed that phiGM22-3 has a genome of 42,662 bp with an identical terminal direct repeat sequence of 328 bp and encodes 58 predicted proteins. Evidence revealed that phiGM22-3 recognized lipopolysaccharides (LPS) as receptor for infection. Additionally, two phage mutants phiMX2 and phiMX8 with different host ranges were identified in the phiGM22-3 population. Phage killing efficiency of P. fluorescens cells artificially inoculated in milk was evaluated. Phage phiGM22-3 and the cocktails containing phiMX2 and phiMX8 can lyse almost 100% bacterial cells at 4°C within 24 h. Taken together, our data indicated that the psychrophilic virus phiGM22-3 and its two mutants can efficiently inhibit bacteria growth at 4°C, showing a great potential to be used as alternatives to conventional antibiotics against P. fluorescens in refrigerated foods.

Identification and characterization of two CRISPR/Cas systems associated with the mosquito microbiome.

The microbiome profoundly influences many traits in medically relevant vectors such as mosquitoes, and a greater functional understanding of host-microbe interactions may be exploited for novel microbial-based approaches to control mosquito-borne disease. Here, we characterized two novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems in Serratia sp. Ag1, which was isolated from the gut of an Anopheles gambiae mosquito. Two distinct CRISPR/Cas systems were identified in Serratia Ag1, CRISPR1 and CRISPR2. Based on cas gene composition, CRISPR1 is classified as a type I-E CRISPR/Cas system and has a single array, CRISPR1. CRISPR2 is a type I-F system with two arrays, CRISPR2.1 and CRISPR2.2. RT-PCR analyses show that all cas genes from both systems are expressed during logarithmic growth in culture media. The direct repeat sequences of CRISPRs 2.1 and 2.2 are identical and found in the arrays of other Serratia spp., including S. marcescens and S. fonticola , whereas CRISPR1 is not. We searched for potential spacer targets and revealed an interesting difference between the two systems: only 9 % of CRISPR1 (type I-E) targets are in phage sequences and 91 % are in plasmid sequences. Conversely, ~66 % of CRISPR2 (type I-F) targets are found within phage genomes. Our results highlight the presence of CRISPR loci in gut-associated bacteria of mosquitoes and indicate interplay between symbionts and invasive mobile genetic elements over evolutionary time.

Studies on ultrasound-mediated insertion-deletion polymorphisms of DNA and underlying mechanisms based on Ames tester strains.

Low-lethality ultrasound technology has received more and more attention in regulating microorganisms of fermentation industry. Herein, two representative Ames tester strains TA97a and TA98 as model organisms were used to explore the effects of ultrasound on insertion-deletion (InDel) polymorphisms of microbial DNA and its underlying mechanisms. Results revealed that a promotion was observed in the reversion mutation of TA98 upon sonication. Sequencing results from 1752 TA98 revertants showed that there was a total of 127 InDels, of which the InDels unique to ultrasound were 36 more than that of the control. Compared with the control, ultrasound-mediated InDels of DNA displayed additional -29 bp deletion and +7∼+43 bp insertions of direct repeat sequences. Combined with the analysis of transcriptomics and prediction of secondary structure of single-stranded DNA from InDels core region (No. 832∼915 bp) in hisD3052 gene of TA98 strain, ultrasound-mediated "thermal breathing" mechanism was proposed based on the formation of DNA hairpin structure with micro-homologous sequence. This finding implied that low-intensity ultrasound is expected to be developed a new low-lethal mutagenic technology for continuous mutagenesis.

Simple and Rapid Non-ribosomal Peptide Synthetase Gene Assembly Using the SEAM-OGAB Method.

Recombination of biosynthetic gene clusters including those of non-ribosomal peptide synthetases (NRPSs) is essential for understanding the mechanisms of biosynthesis. Due to relatively huge gene cluster sizes ranging from 10 to 150 kb, the prevalence of sequence repeats, and inability to clearly define optimal points for manipulation, functional characterization of recombinant NRPSs with maintained activity has been hindered. In this study, we introduce a simple yet rapid approach named "Seamed Express Assembly Method (SEAM)" coupled with Ordered Gene Assembly in Bacillus subtilis (OGAB) to reconstruct fully functional plipastatin NRPS. This approach is enabled by the introduction of restriction enzyme sites as seams at module borders. SEAM-OGAB is then first demonstrated by constructing the ppsABCDE NRPS (38.4 kb) to produce plipastatin, a cyclic decapeptide in B. subtilis. The introduced amino acid level seams do not hinder the NRPS function and enable successful production of plipastatin at a commensurable titer. It is challenging to modify the plipastatin NRPS gene cluster due to the presence of three long direct-repeat sequences; therefore, this study demonstrates that SEAM-OGAB can be readily applied towards the recombination of various NRPSs. Compared to previous NRPS gene assembly methods, the advantage of SEAM-OGAB is that it readily enables the shuffling of NRPS gene modules, and therefore, chimeric NRPSs can be rapidly constructed for the production of novel peptides. This chimeric assembly application of SEAM-OGAB is demonstrated by swapping plipastatin NRPS and surfactin NRPS modules to produce two novel lipopeptides in B. subtilis.

The CRISPR genotypes and genetic diversity of different serogroups of nontyphoidal Salmonella in Guizhou Province, 2013-2018.

Nontyphoidal Salmonella is a bacterial and foodborne pathogen that poses a severe public health threat. However, the genetic diversity of different serogroups of nontyphoidal Salmonella in Guizhou is unknown. This study aimed to obtain the RNA secondary structure of the typical direct repeat sequences, the characteristics of clustered regularly interspaced short palindromic repeats (CRISPR) genotypes, and the genetic diversity of different serogroups of nontyphoidal Salmonella strains. The 342 nontyphoidal Salmonella strains were collected from nine cities (prefectures) of Guizhou province during 2013-2018, serotyped by slide agglutination, and examined the molecular genotypes by CRISPR method. The strains were divided into five serogroups. The dominant serogroup was group B (47.08%), followed by group D1 (36.55%). One hundred and thirty-five CRISPR genotypes were detected with 108 novel spacer sequences amongst 981 unique spacer sequences. The diversity of nontyphoidal Salmonella CRISPR loci was not only the deletion, duplication, or point mutation of spacer sequences but also the acquisition of new spacer sequences to form novel genotypes. The CRISPR genotyping was an effective typing method that could reveal the genetic diversity of different nontyphoidal Salmonella serotypes except for S. Enteritidis.

Biochemical characterization of the two novel mgCas12a proteins from the human gut metagenome

CRISPR/Cas9 and Cas12a belonging to the Class II CRISPR system are characterized by a single-component effector protein. Despite unique features of Cas12a like DNA cleavage with 5′ staggered ends and a single crRNA, Cas12a has not been adopted in biotechnological applications to the similar extent as Cas9. To better understand the CRISPR/Cas12 systems, we selected two candidates, designated mgCas12a-1 and mgCas12a-2, from an analysis of the human microbiome metagenome (mg) and provided biochemical characterization. These new Cas12a proteins shared about 37% identity in amino acid sequences and shared the same direct repeat sequences in the crRNA with FnCas12a from Francisella novicida. The purification yield of the recombinant proteins was up to 3.6-fold greater than that of FnCas12a. In cell-free DNA cleavage assays, both mgCas12a proteins showed the higher cleavage efficiencies when Mn2+ was provided with KCl (< 100 mM) than tested other divalent ions. They were able to tolerate ranges of pH points and temperature, and showed the highest cleavage efficiencies at pH 8.0 and 50 °C. In addition, mgCas12a proteins showed 51% less crRNA-independent and 56% less crRNA-dependent non-specific nuclease activity upon prolonged incubation than did FnCas12a. Considering their greater yield in protein preparation and reduced non-specific nuclease activity, our findings may expedite the use of Cas12a especially when genome editing needs to be practiced with the form of ribonucleoproteins.

Structural basis of direct and inverted DNA sequence repeat recognition by helix-turn-helix transcription factors.

Some transcription factors bind DNA motifs containing direct or inverted sequence repeats. Preference for each of these DNA topologies is dictated by structural constraints. Most prokaryotic regulators form symmetric oligomers, which require operators with a dyad structure. Binding to direct repeats requires breaking the internal symmetry, a property restricted to a few regulators, most of them from the AraC family. The KorA family of transcriptional repressors, involved in plasmid propagation and stability, includes members that form symmetric dimers and recognize inverted repeats. Our structural analyses show that ArdK, a member of this family, can form a symmetric dimer similar to that observed for KorA, yet it binds direct sequence repeats as a non-symmetric dimer. This is possible by the 180° rotation of one of the helix-turn-helix domains. We then probed and confirmed that ArdK shows affinity for an inverted repeat, which, surprisingly, is also recognized by a non-symmetrical dimer. Our results indicate that structural flexibility at different positions in the dimerization interface constrains transcription factors to bind DNA sequences with one of these two alternative DNA topologies.

Genome analysis of Psilogramma increta granulovirus and its intrapopulation diversity

The complete genome of Psilogramma increta granulovirus (PsinGV), isolated from P. increta (Lepidoptera: Sphingidae), was ultra-deep sequenced with a Novaseq PE150 platform and de novo assembled and annotated. The PsinGV genome is a circular double-stranded DNA, 103,721 bp in length, with a G+C content of 33.0%, the third lowest G+C content in present sequenced baculoviruses. It encodes 123 putative open reading frames, including 38 baculovirus core genes, 42 lepidopteran baculovirus conserved genes, 38 betabaculovirus conserved genes, and 5 genes unique to PsinGV. Meanwhile, 3 homologous repeated regions with the core sequence TTGCAA and 3 direct repeated sequences were identified within the PsinGV genome. Kimura two-parameters distance analysis confirmed that Psilogramma increta granulovirus is a representative of a prospective new species of the genus Betabaculovirus. Phylogenetic analysis based on the baculovirus core genes showed that PsinGV is closely related to Choristoneura fumiferana granulovirus, Clostera anastomosis granulovirus-B, and Erinnyis ello granulovirus. These four species therefore share a common ancestor residing in the Betabaculovirus genus. The genome of the PsinGV isolate contained two p10 copies: p10 and p10-2. Phylogenetic reconstruction of P10 suggests a transfer event of the p10-2 gene from an alphabaculovirus to the aforementioned common ancestor. Analysis of genomic diversity showed that 203 intrahost variants, including 182 single nucleotide variants and 21 short insertions/deletions, are present within the PsinGV isolate. Meanwhile, allele frequency indicated that the isolate contains three major genotypes, implying the archived isolate consists of several P. increta carcasses killed by PsinGV with different genotypes.

Analysis of CRISPR-Cas system and antimicrobial resistance in Staphylococcus coagulans isolates.

CRISPR-Cas system contributes adaptive immunity to protect the bacterial and archaeal genome against invading mobile genetic elements. In this study, an attempt was made to characterize the CRISPR-Cas system in Staphylococcus coagulans, the second most prevalent coagulase positive staphylococci causing skin infections in dogs. Out of 45 S. coagulans isolates, 42/45 (93·33%) strains contained CRISPR-Cas system and 45 confirmed CRISPR system was identified in 42 S. coagulans isolates. The length of CRISPR loci ranged from 167 to 2477 bp, and the number of spacers in each CRISPR was varied from two spacers to as high as 37 numbers. Direct repeat (DR) sequences were between 30 and 37, but most (35/45) of the DRs contained 36 sequences. The predominant S. coagulans strains 29/45 did not possess any antimicrobial resistant genes (ARG); 26/29 strains contained Type IIC CRISPR-Cas system. Three isolates from Antarctica seals neither contain CRISPR-Cas system nor ARG. Only 15/45 S. coagulans strains (33·33%) harboured at least one ARG and 13/15 of them were having mecA gene. All the methicillin susceptible S. coagulans isolates contained Type IIC CRISPR-Cas system. In contrast, many (10/13) S. coagulans isolates which were methicillin resistant had Type IIIA CRISPR-Cas system, and this Type IIIA CRISPR-Cas system was present within the SCCmec mobile genetic element. Hence, this study suggests that Type II CRISPR-Cas in S. coagulans isolates might have played a possible role in preventing acquisition of plasmid/phage invasion and Type IIIA CRISPR-Cas system may have an insignificant role in the prevention of horizontal gene transfer of antimicrobial resistance genes in S. coagulans species.

Engineering a self-eliminating transgene in the yellow fever mosquito, Aedes aegypti.

Promising genetics-based approaches are being developed to reduce or prevent the transmission of mosquito-vectored diseases. Less clear is how such transgenes can be removed from the environment, a concern that is particularly relevant for highly invasive gene drive transgenes. Here, we lay the groundwork for a transgene removal system based on single-strand annealing (SSA), a eukaryotic DNA repair mechanism. An SSA-based rescuer strain (kmoRG ) was engineered to have direct repeat sequences (DRs) in the Aedes aegypti kynurenine 3-monooxygenase (kmo) gene flanking the intervening transgenic cargo genes, DsRED and EGFP. Targeted induction of DNA double-strand breaks (DSBs) in the DsRED transgene successfully triggered complete elimination of the entire cargo from the kmoRG strain, restoring the wild-type kmo gene, and thereby, normal eye pigmentation. Our work establishes the framework for strategies to remove transgene sequences during the evaluation and testing of modified strains for genetics-based mosquito control.

Characterization of 67 Confirmed Clustered Regularly Interspaced Short Palindromic Repeats Loci in 52 Strains of Staphylococci

Staphylococcus aureus (S. aureus), which is one of the most important species of Staphylococci, poses a great threat to public health. Clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) are an adaptive immune platform to combat foreign mobile genetic elements (MGEs) such as plasmids and phages. The aim of this study is to describe the distribution and structure of CRISPR-Cas system in S. aureus, and to explore the relationship between CRISPR and horizontal gene transfer (HGT). Here, we analyzed 67 confirmed CRISPR loci and 15 companion Cas proteins in 52 strains of Staphylococci with bioinformatics methods. Comparing with the orphan CRISPR loci in Staphylococci, the strains harboring complete CRISPR-Cas systems contained multiple CRISPR loci, direct repeat sequences (DR) forming stable RNA secondary structures with lower minimum free energy (MFE), and variable spacers with detectable protospacers. In S. aureus, unlike the orphan CRISPRs away from Staphylococcal cassette chromosome mec (SCCmec), the complete CRISPR-Cas systems were in J1 region of SCCmec. In addition, we found a conserved motif 5′-TTCTCGT-3′ that may protect their downstream sequences from DNA interference. In general, orphan CRISPR locus in S. aureus differed greatly from the structural characteristics of the CRISPR-Cas system. Collectively, our results provided new insight into the diversity and characterization of the CRISPR-Cas system in S. aureus.

Antimicrobial Resistance and CRISPR Typing Among Salmonella Isolates From Poultry Farms in China.

Although knowledge of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system has been applied in many research areas, comprehensive studies of this system in Salmonella, particularly in analysis of antibiotic resistance, have not been reported. In this work, 75 Salmonella isolates obtained from broilers or broilers products were characterized to determine their antimicrobial susceptibilities, antibiotic resistance gene profiles, and CRISPR array diversities, and genotyping was explored. In total, 80.00% (60/75) of the strains were multidrug resistant, and the main pattern observed in the isolates was CN-AZM-AMP-AMC-CAZ-CIP-ATM-TE-SXT-FOS-C. The resistance genes of streptomycin (aadA), phenicol (floR-like and catB3-like), β-lactams (blaTEM, blaOXA, and blaCTX), tetracycline [tet(A)-like], and sulfonamides (sul1 and sul2) appeared at higher frequencies among the corresponding resistant isolates. Subsequently, we analyzed the CRISPR arrays and found 517 unique spacer sequences and 31 unique direct repeat sequences. Based on the CRISPR spacer sequences, we developed a novel typing method, CRISPR locus three spacer sequences typing (CLTSST), to help identify sources of Salmonella outbreaks especially correlated with epidemiological data. Compared with multi-locus sequence typing (MLST), conventional CRISPR typing (CCT), and CRISPR locus spacer pair typing (CLSPT), discrimination using CLTSST was weaker than that using CCT but stronger than that using MLST and CLSPT. In addition, we also found that there were no close correlations between CRISPR loci and antibiotics but had close correlations between CRISPR loci and antibiotic resistance genes in Salmonella isolates.

Investigation into the prevalent CRISPR-Cas systems among the Aeromonas genus.

Clustered regularly interspaced short palindromic repeats (CRISPR) is a prokaryotic adaptive immune system that checks invasion by mobile genetic elements through nuclease targeting. In this study, we investigated the occurrence, diversity, and features of the CRISPR system in the genus Aeromonas using bioinformatics tools. Only 13 out of 122 complete genomes (10.66%) of the genus Aeromonas from the NCBI GenBank database harbored the CRISPR system. The Type I-F system was the most prevalent CRISPR system among the Aeromonads, followed by the Type I-E system. Only one strain harbored a Type I-C CRISPR system. Among the Aeromonads, Aeromonas caviae (22.7%) and Aeromonas veronii (20%) had a higher prevalence rate of the complete CRISPR system. The analysis of direct repeat (DR) sequences showed that all could form stable RNA secondary structures. A phylogenetic tree generated for the Cas1 protein classified CRISPR subtypes into three distinct clusters. Among the 748 spacers investigated, 41.98% and 17.25% showed perfect homology to phage and plasmid sequences, respectively. Some arrays had duplicated spacers. The CRISPR loci are closely linked to antibiotic resistance genes in most strains. Collectively, our results would contribute to research on antibiotic resistance in the Aeromonas group, and provide new insights into the diversity and evolution of the CRISPR-Cas system.