Abstract
This protocol describes the isolation of lytic phages infecting the marine bacterium Marinomonas mediterranea from samples of seawater, sand, and seagrass from Posidonia oceanica meadows. It includes the collection of samples, the enrichment method and the isolation and purification of the phages using double layer agar plates. Although the method has been optimized for M. mediterranea, it might be used in the isolation of phages infecting other Marinomonas species and marine bacteria.
Keywords: Marinomonas mediterranea, Isolation of phages from natural samples, Purification of phages, Double layer assay, Phage storage
Background
CRISPR-Cas systems provide adaptive immunity against genetic infection in prokaryotes by acquiring short segments of nucleic acids of the invasive element (spacers). These “molecular memories of infection” are used to produce guide RNAs that target Cas nucleases to the pathogen when a new infection occurs. The spacers are stored directly in the genome of the host, interspersed between directed repeats of the CRISPR arrays. The analysis of the repertoire of spacer sequences in prokaryotic genomes as well as in metagenomic datasets has highlighted a vast diversity of undiscovered genetic pathogens that are the targets of CRISPR-Cas systems (Shmakov et al., 2017). The isolation of phages infecting microorganisms of interest can provide important insights into the mechanism of action of the CRISPR-Cas systems in a natural context.In this work, we provide an easy and inexpensive method for isolating phages infecting the model bacterium Marinomonas mediterranea, from the natural environment of this microorganism. This bacterium has two different CRISPR-Cas systems: a canonical type I-F system, and also one type III-B system capable of acquiring spacers not only from DNA but also from RNA (Silas et al., 2016). This protocol could be easily adapted for the isolation of other phages from marine environments, which contain an enormous diversity of uncharacterized viruses.
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This work has been supported by the grant BFU2017-85464-P (Ministerio de Economía, Industria y Competitividad, Spain). This protocol has been adapted from an “Amplification method” previously described by Suttle (1993). We are thankful to JA García Charton for continuously providing us with the marine samples from the Mediterranean Sea. The authors declare that there are no conflicts of interest.
References
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