A type III-A CRISPR-Cas system employs degradosome nucleases to ensure robust immunity.

Asma Hatoum-Aslan,Lucy Chou-Zheng

doi:10.7554/elife.45393

Abstract

CRISPR-Cas systems provide sequence-specific immunity against phages and mobile genetic elements using CRISPR-associated nucleases guided by short CRISPR RNAs (crRNAs). Type III systems exhibit a robust immune response that can lead to the extinction of a phage population, a feat coordinated by a multi-subunit effector complex that destroys invading DNA and RNA. Here, we demonstrate that a model type III system in Staphylococcus epidermidis relies upon the activities of two degradosome-associated nucleases, PNPase and RNase J2, to mount a successful defense. Genetic, molecular, and biochemical analyses reveal that PNPase promotes crRNA maturation, and both nucleases are required for efficient clearance of phage-derived nucleic acids. Furthermore, functional assays show that RNase J2 is essential for immunity against diverse mobile genetic elements originating from plasmid and phage. Altogether, our observations reveal the evolution of a critical collaboration between two nucleic acid degrading machines which ensures cell survival when faced with phage attack.

Highlights

Most known archaea and about half of bacteria possess adaptive immune systems composed of clusters of regularly-interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins (Makarova et al, 2015)
Our results support a model for CRISPR-Cas10 immunity in which two steps of the pathway, CRISPR RNAs (crRNAs) biogenesis and interference, rely upon the activities of degradosome-associated nucleases, revealing the evolution of a critical collaboration that ensures cell survival when faced with phage infection
We can conclude that while PNPase appears dispensable, RNase J2 is essential for CRISPR immunity against diverse mobile genetic elements originating from plasmid and phage

Summary

Introduction

Most known archaea and about half of bacteria possess adaptive immune systems composed of clusters of regularly-interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins (Makarova et al, 2015). Medical researchers are exploring the use of phages as alternatives to conventional antibiotics and so it is important to find ways to overcome these immune responses in bacteria It remains unclear precisely how Type III CRISPR-Cas systems are able to mount such an effective defense. Previous studies have shown that PNPase and RNase J2 are part of a machine in bacterial cells that usually degrades damaged genetic material These findings show that the Type III CRISPR-Cas system in S. epidermidis has evolved to coordinate with another pathway to help the bacteria survive attack from phages. Staphylococcus epidermidis RP62a harbors a well-established model type III-A CRISPR-Cas system (Figure 1A), here onward referred to as CRISPR-Cas10 This system encodes three spacers (spc) and nine CRISPR-associated proteins (Cas and Csm) that can prevent the transfer of a conjugative plasmid (Marraffini and Sontheimer, 2010) and stave off phage infection (Maniv et al, 2016). In related type III-A systems, target RNA binding has been shown to trigger two additional functions of Cas: the cleavage of non-

B Cas6 Cas6 Cas6

Results

10 J1 J2 dJ1 time

Discussion

Materials and methods