Exploring Streptomyces bacteriophage genomes for CRISPR elements

Introduction: Clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes (CRISPR-Cas) is an acquired immune system that confers resistance in prokaryotes against invading extrachromosomal elements such as viruses and plasmids, and CRISPR editing technology is an important tool in bacterial genetic engineering. Recently, some bacteriophages have been found to harbor CRISPR system elements, suggesting an unexpected reservoir for antiviral genetic elements. Materials and methods: This study examined 325 genomes of Streptomyces bacteriophages for CRISPR elements using CRISPRCasFinder to search for CRISPR arrays and associated cas genes. Putative CRISPR elements were found in 30 genomes, including 11 containing genes identified as coding for Cas4 proteins. These gene products were compared with Clustal Omega (version 1.2.4) and phylogenetic analysis through the neighbor-joining method. Protein homology modeling was carried out using ProMod3, and the phage proteins were aligned to the Cas4 protein from Pyrobaculum calidifontis using the Matchmaker function of UCSF Chimera. Results: The Cas4-like proteins from three groups of Streptomyces phages mapped to the same node as AdnA proteins from Mycolicibacterium smegmatis and Streptomyces coelicolor in the phylogenetic tree analysis. We also found five representative phage protein sequences that contained conserved residues corresponding to functional domains of Cas4 proteins. The modeled protein from Streptomyces phage Circinus showed a moderate to strong alignment and high structural conservation in the core domain to P. calidifontis Cas4 crystal structure. Conclusions: This study utilized bioinformatic and modeling tools to identify likely CRISPR elements in several Streptomyces bacteriophages. These results provide a first step toward understanding how prevalent CRISPR elements are in these bacteriophages and will assist in future experimental studies of possible roles for these elements in phage biology or for use as potential new genetic tools.