Detection and Characterization of Clustered Regularly Interspaced Short Palindromic Repeat-associated Endoribonuclease Gene Variants in Isolated from Seafoods and Environment

Overview

Journal Vet World

Specialty Veterinary Medicine

Date 2019 Jul 23

PMID 31327905

Citations 1

Authors

Pallavi Baliga

Malathi Shekar

Moleyur Nagarajappa Venugopal

Affiliations

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Abstract

Aim: In , the clustered regularly interspaced short palindromic repeat (CRISPR)-associated endoribonuclease gene has been shown to exhibit sequence diversity and has been subtyped into four major types based on its length and composition. In this study, we aimed to detect and characterize the gene variants prevalent among strains isolated from seafoods and environment.

Materials And Methods: Novel primers were designed for each of the subtypes to validate their identification in by polymerase chain reaction (PCR). In total, 38 strains isolated from seafoods and environment were screened for the presence of gene. Few representative PCR products were sequenced, and their phylogenetic relationship was established to available gene sequences in GenBank database.

Results: Of the 38 isolates screened, only about 40% of strains harbored the endoribonuclease gene, among which 31.6% and 7.9% of the isolates were positive for the presence of the 6-a and 6-d subtypes of the gene, respectively. The subtypes -b and -c were absent in strains studied. Sequence and phylogenetic analysis also established the cas6 sequences in this study to match GenBank sequences for -a and d subtypes.

Conclusion: In , the Cas6 endoribonuclease is an associated protein of the CRISPR-cas system. CRISPR-positive strains exhibited genotypic variation for this gene. Primers designed in this study would aid in identifying the genotype and understanding the role of these genotypes in the CRISPR-cas immune system of the pathogen.

Citing Articles

Virulent properties and genomic diversity of isolated from environment, human, diseased fish.

Naknaen A, Surachat K, Manit J, Jetwanna K, Thawonsuwan J, Pomwised R Microbiol Spectr. 2024; 12(7):e0007924.

PMID: 38860819 PMC: 11218479. DOI: 10.1128/spectrum.00079-24.

References

Brouns S, Jore M, Lundgren M, Westra E, Slijkhuis R, Snijders A . Small CRISPR RNAs guide antiviral defense in prokaryotes. Science. 2008; 321(5891):960-4. PMC: 5898235. DOI: 10.1126/science.1159689. View

Horvath P, Barrangou R . CRISPR/Cas, the immune system of bacteria and archaea. Science. 2010; 327(5962):167-70. DOI: 10.1126/science.1179555. View

Haurwitz R, Jinek M, Wiedenheft B, Zhou K, Doudna J . Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science. 2010; 329(5997):1355-8. PMC: 3133607. DOI: 10.1126/science.1192272. View

Carte J, Pfister N, Compton M, Terns R, Terns M . Binding and cleavage of CRISPR RNA by Cas6. RNA. 2010; 16(11):2181-8. PMC: 2957057. DOI: 10.1261/rna.2230110. View

Wang R, Preamplume G, Terns M, Terns R, Li H . Interaction of the Cas6 riboendonuclease with CRISPR RNAs: recognition and cleavage. Structure. 2011; 19(2):257-64. PMC: 3154685. DOI: 10.1016/j.str.2010.11.014. View

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S . MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011; 28(10):2731-9. PMC: 3203626. DOI: 10.1093/molbev/msr121. View

Takeuchi N, Wolf Y, Makarova K, Koonin E . Nature and intensity of selection pressure on CRISPR-associated genes. J Bacteriol. 2011; 194(5):1216-25. PMC: 3294813. DOI: 10.1128/JB.06521-11. View

Wang R, Li H . The mysterious RAMP proteins and their roles in small RNA-based immunity. Protein Sci. 2012; 21(4):463-70. PMC: 3375746. DOI: 10.1002/pro.2044. View

Sternberg S, Haurwitz R, Doudna J . Mechanism of substrate selection by a highly specific CRISPR endoribonuclease. RNA. 2012; 18(4):661-72. PMC: 3312554. DOI: 10.1261/rna.030882.111. View

10.

Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth B, Remm M . Primer3--new capabilities and interfaces. Nucleic Acids Res. 2012; 40(15):e115. PMC: 3424584. DOI: 10.1093/nar/gks596. View

11.

Paranjpye R, Hamel O, Stojanovski A, Liermann M . Genetic diversity of clinical and environmental Vibrio parahaemolyticus strains from the Pacific Northwest. Appl Environ Microbiol. 2012; 78(24):8631-8. PMC: 3502901. DOI: 10.1128/AEM.01531-12. View

12.

Niewoehner O, Jinek M, Doudna J . Evolution of CRISPR RNA recognition and processing by Cas6 endonucleases. Nucleic Acids Res. 2013; 42(2):1341-53. PMC: 3902920. DOI: 10.1093/nar/gkt922. View

13.

Brendel J, Stoll B, Lange S, Sharma K, Lenz C, Stachler A . A complex of Cas proteins 5, 6, and 7 is required for the biogenesis and stability of clustered regularly interspaced short palindromic repeats (crispr)-derived rnas (crrnas) in Haloferax volcanii. J Biol Chem. 2014; 289(10):7164-7177. PMC: 3945376. DOI: 10.1074/jbc.M113.508184. View

14.

Sokolowski R, Graham S, White M . Cas6 specificity and CRISPR RNA loading in a complex CRISPR-Cas system. Nucleic Acids Res. 2014; 42(10):6532-41. PMC: 4041471. DOI: 10.1093/nar/gku308. View

15.

Carte J, Christopher R, Smith J, Olson S, Barrangou R, Moineau S . The three major types of CRISPR-Cas systems function independently in CRISPR RNA biogenesis in Streptococcus thermophilus. Mol Microbiol. 2014; 93(1):98-112. PMC: 4095994. DOI: 10.1111/mmi.12644. View

16.

Sun H, Li Y, Shi X, Lin Y, Qiu Y, Zhang J . Association of CRISPR/Cas evolution with Vibrio parahaemolyticus virulence factors and genotypes. Foodborne Pathog Dis. 2014; 12(1):68-73. DOI: 10.1089/fpd.2014.1792. View

17.

Raghunath P . Roles of thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) in Vibrio parahaemolyticus. Front Microbiol. 2015; 5:805. PMC: 4302984. DOI: 10.3389/fmicb.2014.00805. View

18.

Wang R, Zhong Y, Gu X, Yuan J, Saeed A, Wang S . The pathogenesis, detection, and prevention of Vibrio parahaemolyticus. Front Microbiol. 2015; 6:144. PMC: 4350439. DOI: 10.3389/fmicb.2015.00144. View

19.

Rath D, Amlinger L, Rath A, Lundgren M . The CRISPR-Cas immune system: biology, mechanisms and applications. Biochimie. 2015; 117:119-28. DOI: 10.1016/j.biochi.2015.03.025. View

20.

Charpentier E, Richter H, van der Oost J, White M . Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity. FEMS Microbiol Rev. 2015; 39(3):428-41. PMC: 5965381. DOI: 10.1093/femsre/fuv023. View