Candidate Phyla Radiation Roizmanbacteria From Hot Springs Have Novel and Unexpectedly Abundant CRISPR-Cas Systems.

Lin-Xing Chen,Wen-Jun Li,Basem Al-Shayeb,Jillian F Banfield,Raphaël Méheust,Jennifer A Doudna

doi:10.3389/fmicb.2019.00928

Abstract

The Candidate Phyla Radiation (CPR) comprises a huge group of bacteria that have small genomes that rarely encode CRISPR-Cas systems for phage defense. Consequently, questions remain about their mechanisms of phage resistance and the nature of phage that infect them. The compact CRISPR-CasY system (Cas12d) with potential value in genome editing was first discovered in these organisms. Relatively few CasY sequences have been reported to date, and little is known about the function and activity of these systems in the natural environment. Here, we conducted a genome-resolved metagenomic investigation of hot spring microbiomes and recovered CRISPR systems mostly from Roizmanbacteria that involve CasY proteins that are divergent from published sequences. Within population diversity in the spacer set indicates current in situ diversification of most of the loci. In addition to CasY, some Roizmanbacteria genomes also encode large type I-B and/or III-A systems that, based on spacer targeting, are used in phage defense. CRISPR targeting identified three phage represented by complete genomes and a prophage, which are the first reported for bacteria of the Microgenomates superphylum. Interestingly, one phage encodes a Cas4-like protein, a scenario that has been suggested to drive acquisition of self-targeting spacers. Consistent with this, the Roizmanbacteria population that it infects has a CRISPR locus that includes self-targeting spacers and a fragmented CasY gene (fCasY). Despite gene fragmentation, the PAM sequence is the same as that of other CasY reported in this study. Fragmentation of CasY may avoid the lethality of self-targeting spacers. However, the spacers may still have some biological role, possibly in genome regulation. The findings expand our understanding of CasY diversity, and more broadly, CRISPR-Cas systems and phage of CPR bacteria.

Highlights

The Candidate Phyla Radiation (CPR) comprises a huge fraction of Domain Bacteria
Candidate Phyla Radiation bacteria account for a huge amount of diversity within the Bacterial domain, but the mechanisms of their interactions with phage and the phage that infect them have remained largely undocumented
We report an unexpected diversity of CRISPR-Cas systems in the genomes of bacteria from the CPR phylum of Roizmanbacteria, both from newly reconstructed sequences from multiple hot spring sediments of Tibet, China (Supplementary Table S1) and some previously published genomes

Summary

Introduction

The Candidate Phyla Radiation (CPR) comprises a huge fraction of Domain Bacteria. The CPR bacteria uniformly have small genomes (often ∼1 Mbp) and limited biosynthetic capacity (Brown et al, 2015; Anantharaman et al, 2016; Hug et al, 2016; Castelle and Banfield, 2018). A previous meta-analysis found that only 2.4% of organisms from the Parcubacteria (OD1) and Microgenomates (OP11) superphyla encode CRISPR-Cas systems in their genomes, as compared to 47.4% in archaea and 24.4% in non-CPR bacteria (Burstein et al, 2016). The authors noted that when CRISPR-Cas systems occur in CPR bacteria they tend to be different from those found in other bacteria. Four genomes from Dojkabacteria (WS6), Parcubacteria (OD1) and Roizmanbacteria were previously recognized to encode CRISPRCas12a (Cpf1) systems (Zetsche et al, 2015), and more recently, six genomes were reported encoding a newly recognized compact CasY effector enzyme that has genome editing potential (Burstein et al, 2017)

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Results

Conclusion