Clustered Regularly Interspaced Short Palindromic Repeats Loci Research Articles

Clustered Regularly Interspaced Short Palindromic Repeat-1 (CRISPR-1) Locus as a Tool for Tracing the Zoonotic History of Salmonella enterica Strains.

Background Salmonella enterica is a significant foodborne pathogen that causes considerable illness and death in humans and animals. The clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) system in bacteria acts as an adaptive immune defense against invasive genetic elements by incorporating short intergenic spacers (IGSs) into CRISPR loci. These loci serve as molecular records of past interactions with phages and plasmids, providing insights into the transmission and evolution of bacterial strains across different hosts. Aim This study aimed to investigate the diversity of IGSs in the CRISPR-1 locus of S. enterica isolates from humans and camels. The objective was to assess the potential of IGSs to distinguish strains, track sources, and understand patterns of zoonotic transmission. Materials and methods Genomic DNA was extracted from multiple strains of S. enterica, and the CRISPR-1 locus was polymerase chain reaction (PCR) amplified and sequenced. The sequences were compared to identify distinct patterns of IGSs and potential host-specific characteristics. Sanger sequencing and bioinformatics tools were used to classify the IGSs and determine their similarity to known sequences in the National Center for Biotechnology Information (NCBI) database. Results Sequence analysis revealed five distinct CRISPR-1 types among S. enterica isolates from humans and three among camel isolates. The presence of shared IGSs between human and camel S. entericaisolates suggested zoonotic or reverse-zoonotic transmission events. Additionally, host-specific unknown IGSs (UIGS) were identified. Importantly, camel isolates initially identified as S. enterica subspeciesenterica serovar Enteritidis based on rrnH gene sequencing were reclassified as S. enterica serovar Enteritidis based on CRISPR-1 profiling, demonstrating the higher resolution of CRISPR-based genotyping. Conclusion The diversity of IGSs in the CRISPR-1 locus effectively differentiated S. enterica strains and provided insights into their evolutionary origins and transmission dynamics. CRISPR-based genotyping proves to be a promising tool to complement traditional serotyping methods, enhancing the molecular epidemiology of salmonellosis and potentially leading to better management and control strategies for this pathogen.

Molecular and bacteriological investigations for the co-existence CRISPR/Cas system and β-lactamases of types extended-spectrum and carbapenemases in Multidrug, extensive drug and Pandrug-Resistant Klebsiella pneumoniae

The recent approach towards combating the antimicrobial resistance has led to the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated sequence to overcome the challenges of antimicrobial resistance. Thus, this study aimed to detect the underlying resistance mechanisms such as ESBLs and carbapenemases and whether there is a correlation between multidrug, extensive drug and pan drug resistance and the occurrence of CRISPR loci. A total of one hundred study isolates were subjected to antimicrobial susceptibility testing using the AST card of the Vitek technique to detect resistance patterns involving ESBLs and carbapenemase (CRE). An investigation of the genes encoding CRISPR/Cas systems using PCR was achieved. Out of 81 (81.0%) resistant Klebsiella pneumoniae isolates, 71 (71%) and 21 (21.0%) produced ESBLs and carbapenemases, respectively. Also, 53 (53.0%), 19 (19.0%) and 9 (9.0%) were MDR, XDR, and PDR respectively. It was noted that Cas1, Cas3, CRISPR1, CRISPR2 and CRISPR3 were positive in 38 (38.0%) of the isolates, while CRISPR1 for incomplete CRISPR1-Cas systems alone was detected in 78 (78.0%). Further, the number of intact CRISPR1, intact CRISPR2 and intact CRISPR3 types were 7 (27.0%), 34 (34%) and 18 (18.0%) respectively. It is concluded that antibiotic resistance levels were inversely correlated with the existence of CRISPR/Cas systems. The absence of the CRISPR/Cas system increases the prevalence of MDR, XDR and PDR in ESBL and carbapenem-producing Klebsiella pneumoniae. With the increase in the degree of antibiotic resistance (MDR, XDR to PDR), the occurrence ratio of the (CRISPR)/CRISPR-associated sequence decreased.

Exploring the Interplay of the CRISPR-CAS System with Antibiotic Resistance in Staphylococcus aureus: A Poultry Meat Study from Lahore, Pakistan.

Staphylococcus aureus is one of the major pathogens responsible for causing food poisoning worldwide. The emergence of antibiotic resistance in this bacterium is influenced by various factors. Among them, bacterial acquired defense systems described as clustered regularly interspaced short palindromic repeats (CRISPR)-cas system might be involved in antibiotic resistance development in bacteria. The current study was designed to assess the prevalence of S. aureus and its antibiotic resistance profile and identify the relationship of the CRISPR-cas system with antimicrobial resistance, followed by phylogenetic analysis. Total samples (n = 188) of poultry meat were collected from the poultry bird market of Lahore, Punjab, Pakistan. We used both phenotypic (antibiotic disc diffusion) and genotypic methods (PCR) to identify multi-drug resistant (MDR) strains of S. aureus. Additionally, the role of the CRISPR-Cas system in the isolated MDR S. aureus was also assessed. In addition, real-time quantitative PCR (qRT-PCR) was used to evaluate the association of the CRISPR-cas system with antimicrobial resistance. All of the S. aureus isolates showed 100% resistance against erythromycin, 97.5% were resistant to tetracycline, and 75% were resistant to methicillin. Eleven isolates were MDR in the current study. The CRISPR system was found in all MDR isolates, and fifteen spacers were identified within the CRISPR locus. Furthermore, MDR S. aureus isolates and the standard strain showed higher expression levels of CRISPR-associated genes. The correlation of said system with MDR isolates points to foreign gene acquisition by horizontal transfer. Current knowledge could be utilized to tackle antibiotic-resistant bacteria, mainly S. aureus.

CRISPR-Cas systems feature and targeting phages diversity in Lacticaseibacillus rhamnosus strains.

One of the most important adaptive immune systems in bacteria against phages is clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CAS) genes. In this investigation, an approach based on genome mining was employed to characterize the CRISPR-Cas systems of Lacticaseibacillus rhamnosus strains. The analysis involved retrieving complete genome sequences of L. rhamnosus strains, and assessing the diversity, prevalence, and evolution of their CRISPR-Cas systems. Following this, an analysis of homology in spacer sequences from identified CRISPR arrays was carried out to investigate and characterize the range of target phages. The findings revealed that 106 strains possessed valid CRISPR-Cas structures (comprising CRISPR loci and Cas genes), constituting 45% of the examined L. rhamnosus strains. The diversity observed in the CRISPR-Cas systems indicated that all identified systems belonged to subtype II-A. Analyzing the homology of spacer sequences with phage and prophage genomes discovered that strains possessing only CRISPR-Cas subtype II targeted a broader spectrum of foreign phages. In summary, this study suggests that while there is not significant diversity among the CRISPR-Cas systems identified in L. rhamnosus strains, there exists notable variation in subtype II-A systems between L. rhamnosus and other lactobacilli. The diverse nature of these CRISPR-Cas systems underscores their natural activity and importance in adaptive immunity.

CRISPR-Cas systems and diversity of targeting phages in Lactobacillus johnsonii strains; insights from genome mining approach.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CAS) genes make up bacteria's adaptive immune system. These genes protect bacteria from being eaten by bacteriophages. In this study, CRISPR-Cas systems were characterized using a genomic approach. For this purpose, genome sequences of Lactobacillus johnsonii strains were retrieved, and the diversity, occurrence, and evolution of the CRISPR-Cas systems were analyzed. Then, homology analyses of spacer sequences in identified CRISPR arrays were performed to analyze and characterize the diversity of target phages and plasmids. Finally, the evolutionary paths of spaceromes in each subtype of CRISPR arrays were performed using acquisition and deletion events surveyed under the selective pressure of foreign plasmids and phages. Results showed that 138 strains contain valid CRISPR-Cas structures (CRISPR loci together with the Cas genes) in their genomes, which accounted for about 17% of the L. johnsonii studied strains belonging to subtypes II-A, I-E, and I-C. Moreover, results indicated that some specific groups of plasmids were targeted with specific CRISPR array systems. Homology analysis of spacer sequences with phage genomes also revealed that spacers of strains that harbored only CRISPR-Cas subtype-II targeted a greater diversity of foreign phages. In conclusion, the current study indicates that there is great diversity between the CRISPR-Cas systems identified in L. johnsonii strains. Such diverse CRISPR-Cas systems indicate that these systems are naturally active and important in terms of adaptive immunity and evolutionary relationships.

Comparison of Structural Features of CRISPR-Cas Systems in Thermophilic Bacteria.

The clustered regularly interspaced short palindromic repeat (CRISPR) is an adaptive immune system that defends most archaea and many bacteria from foreign DNA, such as phages, viruses, and plasmids. The link between the CRISPR-Cas system and the optimum growth temperature of thermophilic bacteria remains unclear. To investigate the relationship between the structural characteristics, diversity, and distribution properties of the CRISPR-Cas system and the optimum growth temperature in thermophilic bacteria, genomes of 61 species of thermophilic bacteria with complete genome sequences were downloaded from GenBank in this study. We used CRISPRFinder to extensively study CRISPR structures and CRISPR-associated genes (cas) from thermophilic bacteria. We statistically analyzed the association between the CRISPR-Cas system and the optimum growth temperature of thermophilic bacteria. The results revealed that 59 strains of 61 thermophilic bacteria had at least one CRISPR locus, accounting for 96.72% of the total. Additionally, a total of 362 CRISPR loci, 209 entirely distinct repetitive sequences, 131 cas genes, and 7744 spacer sequences were discovered. The average number of CRISPR loci and the average minimum free energy (MFE) of the RNA secondary structure of repeat sequences were positively correlated with temperature whereas the average length of CRISPR loci and the average number of spacers were negatively correlated. The temperature did not affect the average number of CRISPR loci, the average length of repeats, or the guanine-cytosine (GC) content of repeats. The average number of CRISPR loci, the average length of the repeats, and the GC content of the repeats did not reflect temperature dependence. This study may provide a new basis for the study of the thermophilic bacterial adaptation mechanisms of thermophilic bacteria.

Characterization and diversity of CRISPR/Cas systems in Klebsiella oxytoca.

CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) system is a crucial adaptive immune system for bacteria to resist foreign DNA infection. In this study, we investigated the prevalence and diversity of CRISPR/Cas systems in 175 Klebsiella oxytoca (K. oxytoca) strains. Specifically, 58.86% (103/175) of these strains possessed at least one confirmed CRISPR locus. Two CRISPR/Cas system types, I-F and IV-A3, were identified in 69 strains. Type I-F system was the most prevalent in this species, which correlated well with MLST. Differently, type IV-A3 system was randomly distributed. Moreover, the type IV-A3 system was separated into two subgroups, with subgroup-specific cas genes and repeat sequences. In addition, spacer origin analysis revealed that approximately one-fifth of type I-F spacers and one-third of type IV-A3 spacers had a significant match to MGEs. The phage tail tape measure protein and conjunctive transfer system protein were important targets of type I-F and IV-A3 systems in K. oxytoca, respectively. PAM sequences were inferred to be 5'-NCC-3' for type I-F, 5'-AAG-3' for subgroup IV-A3-a, and 5'-AAN-3' for subgroup IV-A3-b. Collectively, our findings will shed light on the prevalence, diversity, and functional effects of the CRISPR/Cas system in K. oxytoca.

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.

Diversity of CRISPR-Cas type II-A systems in Streptococcus anginosus.

Streptococcus anginosus is a commensal Streptococcal species that is often associated with invasive bacterial infections. However, little is known about its molecular genetic background. Many Streptococcal species, including S. anginosus, harbor clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems. A CRISPR-Cas type II-A system as well as a type II-C system have been reported for this species. To characterize the CRISPR-Cas type II systems of S. anginosus in more detail, we conducted a phylogenetic analysis of Cas9 sequences from CRISPR-Cas type II systems with a special focus on streptococci and S. anginosus. In addition, a phylogenetic analysis of S. anginosus strains based on housekeeping genes included in MLST analysis, was performed. All analyzed Cas9 sequences of S. anginosus clustered with the Cas9 sequences of CRISPR type II-A systems, including the Cas9 sequences of S. anginosus strains reported to harbor a type II-C system. The Cas9 genes of the CRISPR-Cas type II-C systems of other bacterial species separated into a different cluster. Moreover, analyzing the CRISPR loci found in S. anginosus, two distinct csn2 genes could be detected, a short form showing high similarity to the canonical form of the csn2 gene present in S. pyogenes. The second CRISPR type II locus of S. anginosus contained a longer variant of csn2 with close similarities to a csn2 gene that has previously been described in Streptococcus thermophilus. Since CRISPR-Cas type II-C systems do not contain a csn2 gene, the S. anginosus strains reported to have a CRISPR-Cas type II-C system appear to carry a variation of CRISPR-Cas type II-A harboring a long variant of csn2.

Analysis of genetic structure and function of clustered regularly interspaced short palindromic repeats loci in 110 Enterococcus strains.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) are an adaptive immune system involved in specific defenses against the invasion of foreign mobile genetic elements, such as plasmids and phages. This study aims to analyze the gene structure and to explore the function of the CRISPR system in the Enterococcus genome, especially with regard to drug resistance. The whole genome information of 110 enterococci was downloaded from the NCBI database to analyze the distribution and the structure of the CRISPR-Cas system including the Cas gene, repeat sequences, and spacer sequence of the CRISPR-Cas system by bioinformatics methods, and to find drug resistance-related genes and analyze the relationship between them and the CRISPR-Cas system. Multilocus sequence typing (MLST) of enterococci was performed against the reference MLST database. Information on the drug resistance of Enterococcus was retrieved from the CARD database, and its relationship to the presence or absence of CRISPR was statistically analyzed. Among the 110 Enterococcus strains, 39 strains (35.45%) contained a complete CRISPR-Cas system, 87 CRISPR arrays were identified, and 62 strains contained Cas gene clusters. The CRISPR system in the Enterococcus genome was mainly type II-A (59.68%), followed by type II-C (33.87%). The phylogenetic analysis of the cas1 gene sequence was basically consistent with the typing of the CRISPR-Cas system. Of the 74 strains included in the study for MLST typing, only 19 (25.68%) were related to CRISPR-Cas typing, while the majority of the strains (74.32%) of MLST typing were associated with the untyped CRISPR system. Additionally, the CRISPR-Cas system may only be related to the carrying rate of some drug-resistant genes and the drug-resistant phenotype. In conclusion, the distribution of the enterococcus CRISPR-Cas system varies greatly among different species and the presence of CRISPR loci reduces the horizontal transfer of some drug resistance genes.

Clarifying CRISPR: Why Repeats Identified in the Human Genome Should Not Be Considered CRISPRs.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated genes (cas) are essential components of adaptive immune systems that protect bacteria and archaea from viral infection. CRISPR-Cas systems are found in about 40% of bacterial and 85% of archaeal genomes, but not in eukaryotic genomes. Recently, an article published in Communications Biology reported the identification of 12,572 putative CRISPRs in the human genome, which they call "hCRISPR." In this study, we attempt to reproduce this analysis and show that repetitive elements identified as putative CRISPR loci in the human genome contain neither the repeat-spacer-repeat architecture nor the cas genes characteristic of functional CRISPR systems.

Prevalence and Characterization of CRISPR Locus 2.1 Spacers in Escherichia coli Isolates Obtained from Feces of Animals and Humans.

The clustered regularly interspaced short palindromic repeat (CRISPR) has been studied as an immune system in prokaryotes for the survival of bacteriophages. The CRISPR system in prokaryotes records the invasion of bacteriophages or other genetic materials in CRISPR loci. Accordingly, CRISPR loci can reveal a history of infection records of bacteriophages and other genetic materials. Therefore, identification of the CRISPR array may help trace the events that bacteria have undergone. In this study, we characterized and identified the spacers of the CRISPR loci in Escherichia coli isolates obtained from the feces of animals and humans. Most CRISPR spacers were found to stem from phages. Although we did not find any patterns in CRISPR spacers according to sources, our results showed that phage-derived spacers mainly originated from the families Inoviridae, Myoviridae, Podoviridae, and Siphoviridae and the order Caudovirales, whereas plasmid-derived CRISPR spacers were mainly from the Enterobacteriaceae family. In addition, it is worth noting that the isolates from each animal and human source harbored source-specific spacers. Considering that some of these taxa are likely found in the gut of mammalian animals, CRISPR spacers identified in these E. coli isolates were likely derived from the bacteriophageome and microbiome in closed gut environments. Although the bacteriophageome database limits the characterization of CRISPR arrays, the present study showed that some spacers were specifically found in both animal and human sources. Thus, this finding may suggest the possible use of E. coli CRISPR spacers as a microbial source tracking tool. IMPORTANCE We characterized spacers of CRISPR locus 2.1 in E. coli isolates obtained from the feces of various sources. Phage-derived CRISPR spacers are mainly acquired from the order Caudovirales, and plasmid-derived CRISPR spacers are mostly from the Enterobacteriaceae family. This is thought to reflect the microbiome and phageome of the gut environment of the sources. Hence, spacers may help track the encounter of bacterial cells with bacterial cells, viruses, or other genetic materials. Interestingly, source-specific spacers are also observed. The identification of source-specific spacers is thought to help develop the methodology of microbial source tracking and understanding the interactions between viruses and bacteria. However, very few spacers have been uncovered to track where they originate. The accumulation of genome sequences can help identify the hosts of spacers and can be applied for microbial source tracking.

Comparative genomics reveals the diversity of CRISPR-Cas locus in Azotobacter organisms.

Clustered regularly interspaced short palindromic repeats (CRISPRs) are known to provide adaptive immunity to bacteria against invading bacteriophages. In recent years, CRISPR-based technologies have been used for creating improved plant varieties; however, the indigenous CRISPR-Cas elements of plant growth-promoting bacteria are usually neglected. These indigenous genetic cassettes have evolved over millions of years and have shaped the bacterial genome. Therefore, these genetic loci can be used to study the adaptive capability of the bacteria in the environment. This study aims to bioinformatically analyze the genomes of a common free-living nitrogen-fixing Azotobacter spp. to assess their CRISPR-Cas diversity. Strains of Azotobacter vinelandii and Azotobacter chroococcum were found to harbor a large number of spacers. The phylogeny of different Cas and Cse1 proteins revealed a close evolutionary relationship among A. chroococcum B3, A. chroococcum NCIMB 8003 locus II, and A. vinelandii DJ locus I. The secondary structure of the hairpin loop of the repeat was also analyzed, and a correlation was derived between the structural stability of the hairpin loop and the number of spacers acquired by the CRISPR loci. These findings revealed the diversity and evolution of the CRISPR sequences and Cas proteins in Azotobacter species. Although the adaptive immune system of bacteria against bacteriophage, CRISPR-Cas, has been identified in many bacteria, studies of plant growth-promoting bacteria have been neglected. These indigenous CRISPRs have shaped the genome over millions of years and their study can lead to the understanding of the genome composition of these organisms. Our results strengthen the idea of using A. chroococcum and A. vinelandii as biofertilizer strains as they possess more spacers with highly stable repeat sequences, thereby imparting them higher chance of survival against mobile genetic elements like phages and plasmids.

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.

Clustered Regularly Interspaced Short Palindromic Repeats in Xanthomonas citri-Witnesses to a Global Expansion of a Bacterial Pathogen over Time.

Xanthomonas citri pv. citri, a Gram-negative bacterium, is the causal agent of citrus canker, a significant threat to citrus production. Understanding of global expansion of the pathogen and monitoring introduction into new regions are of interest for integrated disease management at the local and global level. Genetic diversity can be assessed using genomic approaches or information from partial gene sequences, satellite markers or clustered regularly interspaced short palindromic repeats (CRISPR). Here, we compared CRISPR loci from 355 strains of X. citri pv. citri, including a sample from ancient DNA, and generated the genealogy of the spoligotypes, i.e., the absence/presence patterns of CRISPR spacers. We identified 26 novel spoligotypes and constructed their likely evolutionary trajectory based on the whole-genome information. Moreover, we analyzed ~30 additional pathovars of X. citri and found that the oldest part of the CRISPR array was present in the ancestor of several pathovars of X. citri. This work presents a framework for further analyses of CRISPR loci and allows drawing conclusions about the global spread of the citrus canker pathogen, as exemplified by two introductions in West Africa.

Genomic features of a new head–tail halovirus VOLN27B infecting a Halorubrum strain

Relatively few viruses infecting haloarchaea (haloviruses) have been reported. In this study, the genome sequence of VOLN27B, a recently described archaeal tailed virus (arTV) with a myovirus morphotype was described, along with the sequence of its host, Halorubrum spp. LN27. Halovirus VOLN27B contains a linear, dsDNA genome of 76,891 bp which is predicted to encode 109 proteins and four tRNAs (tRNAThr, tRNAArg, tRNAGly and tRNAAsn). The DNA G + C content of VOLN27B genome is 56.1 mol%, nearly 10% lower than that of its host strain. A 315 bp LTR (long terminal repeat) was detected in the genome. The genome of its host strain LN27 was 3,301,211 bp (chromosome and 1 plasmid) with a DNA G + C content of 68.3 mol% and 3142 annotated protein coding genes. At least two hypothetical proviruses were detected in the genome. It lacked a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) locus. Sequence similarity and phylogenetic tree reconstructions placed it within the genus Halorubrum as a potential new species. VOLN27B exhibits a distinct difference in the frequency of codon usage against its host strain Halorubrum sp. LN27. The organization of VOLN27B genome shows remarkable synteny and amino acid sequence similarity to the genomes and predicted proteins of HF1-like haloviruses (genus Haloferacalesvirus) and a provirus in the genome of Halorubrum depositum Y78. VOLN27B and its host Halorubrum sp. LN27 comprise a new virus-host system from a hypersaline ecosystem and can be used to further understand the novel biology at extreme salt concentration.

Comparison of Propionibacterium genomes: CRISPR-Cas systems, phage/plasmid diversity, and insertion sequences.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems constitute the adaptive immune system in prokaryotes that provide resistance against invasive genetic elements. The genus Propionibacterium comprises gram-positive, facultative anaerobe, non-spore-forming bacteria, and is the source of some B group vitamins such as B12 as well as bacteriocins. Some of the selected species of the genus Propionibacterium spp. were reclassified into the three genera in 2016 (Acidipropionibacterium spp., Pseudopropionibacterium spp., Cutibacterium spp.). Therefore, this study compared CRISPR/Cas systems, Cas 1 and repeat sequences phylogeny, phage/plasmid surveys as well as insertion sequences of new genera members. In this study, a total of 34 genomes of 13 species were observed with a bioinformatic approach. CRISPR-Cas + + and CRISPRDetect were used to detect CRISPR/Cas systems, direct repeats, and spacers. 39 CRISPR-Cas systems were detected. Type I-E, Type I-U, and one incomplete III-B CRISPR-Cas subtypes were identified. Most of the strains had Cas1/Cas4 fusion proteins. Pseudopropionibacterium propionicum strains had two types I-U and one of the CRISPR loci had csx17 cas genes. Common phage invaders were Propionibacterium phage E6, G4, E1, Anatole, and Doucette. The BLSM62 similarity score of all Cas1 sequences was 48.4% while the pairwise identity of repeat sequences was 48.7%. Common insertion sequences were ISL3, IS3, IS30. The diversity analysis of the CRISPR/Cas system in the genus Propionibacterium provided a new perspective for determining the role of the CRISPR-Cas system in the evolution of new genera.

Genome Mining Approach Reveals the Occurrence and Diversity Pattern of Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated Systems in Lactobacillus brevis Strains.

Clustered regularly interspaced short palindromic repeats (CRISPR) together with their CRISPR-associated (Cas) genes are widely distributed in prokaryotes that provide an adaptive defense mechanism against foreign invasive DNA. There is relatively little knowledge about the CRISPR-Cas diversity and evolution in Lactobacillus brevis strains. Therefore, in this study, a genome-mining approach was employed to investigate the diversity and occurrence of the CRISPR-Cas system in 83 L. brevis strains. Moreover, trans-activating CRISPR RNA (tracrRNA) and protospacer adjacent motif (PAM) as pivotal elements for the successful targeting and inference of phages by the subtype II CRISPR-Cas systems were surveyed. Finally, evolutionary paths of L. brevis strains under selective pressure from foreign invasive DNA such as plasmids and phages of studied strains were surveyed using acquisition and deletion events analysis of spacers. A total of 127 confirmed CRISPRs were identified, which were distributed in 69 strains. Among strains with confirmed CRISPRs, 35 strains only contained one CRISPR locus, 23 strains contained two CRISPR loci, and 12 strains contained three to six CRISPR loci. L. brevis strains frequently harbor more than one CRISPR system. Analysis of confirmed CRISPR arrays showed that 31 out of 127 confirmed CRISPRs included Cas genes which were categorized as one of the II-A, II-C, and I-E subtypes. Analysis of subtype II-A spacers reflected divergent evolution for 18 strains into 16 unique groups. Additional analysis of spacer sequences also confirmed the implication of characterizing CRISPR-Cas systems in targeting of phages and plasmids. The current study highlighted the potential of utilizing CRISPR spacer polymorphism in genotyping lactobacillus strains. Moreover, it provides deep insights into the occurrence, diversity, and functional impacts of the CRISPR-Cas system in L. brevis strains.

Characterization of Xanthomonas citri pv. citri from China based on spoligotyping

Xanthomonas citri pv. citri (Xcc), a gram-negative bacterium, is the causal agent of citrus canker, one of the most devastating diseases threatening the citrus industry worldwide. Understanding the diversity and population structure of Xcc is a prerequisite for disease epidemiological monitoring and effective disease management. Recent characterization of the clustered regularly interspaced short palindromic repeats (CRISPR)/cas (CRISPR-associated proteins genes) system with a highly variable repeat number among species provides a new molecular typing method for bacterial genetic analysis. In this study, we performed systematic in silico analyses of 28 Xcc genomes and identified a credible CRISPR/cas in Xcc strains. Further analysis of CRISPR polymorphisms (repeat number and spacer types) in 129 XccA strains collected from six provinces in China identified 15 types of CRISPR arrays with 25 spacers. Phylogenetic analysis of Xcc strains based on the CRISPR locus produced a more reliable and accurate typing result compared to the commonly used loci. In addition, seven associated cas genes—cas1, cas2, cas3, cas4, cas5, cas7 (csd2), and cas8 (csd1)—were found located adjacent to the CRISPR array. BLAST results showed > 99% similarity of seven cas genes among Xcc strains. Homology analysis of spacer sequences showed that six spacers had possible phage/prophage origin. The characterization of the CRISPR/cas system among Xcc strains provided an updated strain typing method for Xcc diversity analysis and yielded a panoramic view of CRISPR evolution for further studies of Xcc-phage interactions.

Genotyping Based on CRISPR Loci Diversity and Pathogenic Potential of Diarrheagenic Escherichia coli.

Diarrheagenic Escherichia coli (DEC) can cause epidemic diarrhea worldwide. The pathogenic potential of different strains is diverse and the continuous emergence of pathogenic strains has brought serious harm to public health. Accurately distinguishing and identifying DEC with different virulence is necessary for epidemiological surveillance and investigation. Clustered regularly interspaced short palindromic repeats (CRISPR) typing is a new molecular method that can distinguish pathogenic bacteria excellently and has shown great promise in DEC typing. The purpose of this study was to investigate the discrimination of CRISPR typing method for DEC and explore the pathogenicity potential of DEC based on CRISPR types (CT). The whole genome sequences of 789 DEC strains downloaded from the database were applied CRISPR typing and serotyping. The D value (Simpson’s index) with 0.9709 determined that CRISPR typing had a higher discrimination. Moreover, the same H antigen strains with different O seemed to share more identical spacers. Further analyzing the strains CRISPR types and the number of virulence genes, it was found that there was a significant correlation between the CRISPR types and the number of virulence genes (p < 0.01). The strains with the largest number of virulence genes concentrated in CT25 and CT56 and the number of virulence genes in CT264 was the least, indicating that the pathway potential of different CRISPR types was variable. Combined with the Caco-2 cell assay of the laboratory strains, the invasion capacity of STEC strains of different CRISPR types was different and there was no significant difference in the invasion rate between different CRISPR type strains (p > 0.05). In the future, with the increase of the number of strains that can be studied experimentally, the relationship between CRISPR types and adhesion and invasion capacities will be further clarified.