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. 2019 May 3;10:928.
doi: 10.3389/fmicb.2019.00928. eCollection 2019.

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

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Free PMC article

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

Lin-Xing Chen et al. Front Microbiol. .
Free PMC article

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.

Keywords: CPR; CRISPR-Cas; Roizmanbacteria; hot spring; phage.

Figures

FIGURE 1
FIGURE 1
Roizmanbacteria and Woesebacteria genomes encode CRISPR-CasY and/or other CRISPR-Cas systems. (A) CRISPR systems detected in genomes of CPR bacteria (top left), the phylogenetic classification of which was established based on concatenated sequences of 16 ribosomal proteins (top right). Included in the analyses are the dereplicated representatives of Roizmanbacteria and Woesebacteria genomes from this study (in red) and Roizmanbacteria genomes from NCBI (in black). CRISPR system types in each genome are indicated by the symbols after the genome names, and the number of non-redundant genomes is shown in brackets. Those clades without CRISPR-Cas are collapsed, and the number of genomes are shown (see Supplementary Figure S1 for the uncollapsed tree). The red arrow indicates the presence of a restriction-modification system, the hypothetical proteins are shown in white. (B) Phylogenetic analyses of CasY proteins, including those previously reported and those identified in this study. The local alignment of conserved motifs of CasY protein, including RuvC-I, -II, -III and helical, are shown. The catalytic residues are shown by white letters on a black background; for other residues, backgrounds of different colors are used if the amino acids are inconsistent among those CasY identified in this study.
FIGURE 2
FIGURE 2
Prophage and restriction modification systems are detected in the genomes of Roizmanbacteria C2-Gp3. (A) Scaffold 44 includes prophage, a restriction-modification system and an apparently degenerate Type III-A CRISPR-Cas system. (B) The proteins in the restriction-modification system shown in (A). (C) Prophage genes targeted by spacers are shown in black and the number of spacers targeting each open reading frame (ORF) is listed in brackets following the annotation. Genes not targeted by CRISPR spacers are shown in orange (top panel). The genome affiliations of spacers targeting the prophage are indicated in the bottom panel.
FIGURE 3
FIGURE 3
Phylogeny of capsid proteins used for taxonomic assignment of Roizmanbacteria-infecting phage (or prophage) in this study. The Roizmanbacteria-infecting phage and prophage are shown in red, and those with spacer targets are indicated by triangles. Squares indicate phage determined to be similar based on their capsid protein sequences. The previously reported Absconditabacteria (SR1) phage are included for comparison.
FIGURE 4
FIGURE 4
Complete genomes of Roizmanbacteria-infecting phage. The red rings represent (A) QZM_A2_Phage_33_19 and (B) QZM_B3_Phage_33_79 phage genomes. The ORFs are shown outside the genomes, those targeted by at least one spacer are in black (genes not targeted are in orange). The total number of spacers that target each gene is listed in parentheses following the protein annotation. The spacers targeting the phage genome from a given CRISPR-Cas system are indicated by bars on the dotted inner rings (see Figure 1 for CRISPR-Cas system type). Bars are colored by genome of origin (see top right). The non-shared proteins between these two phage genomes are indicated by green circles and numbered, their annotations are shown at the right. Hyp, hypothetical protein. (C) The coverage information of these two phage genomes in QZM-related samples.
FIGURE 5
FIGURE 5
One Roizmanbacteria genome encodes and unusual CRISPR system with a fragmented CasY (fCasY) protein and self-targeting spacers. (A) Mutations leading to fragmentation of CasY proteins into three pieces (red arrows) and their incidence in the population, and other features of the locus. (B) The reconstructed CRISPR locus showing the history of spacer acquisition and the distribution of self-targeted spacers (marked by red circles). (C) Scaffolds encoding genes and an intergenic region matching the self-targeting spacers. The targeted genes have the same color as the corresponding spacers in (B), genes targeted by single copy spacers [white in (B)] are indicated by numbers, and CRISPR-Cas systems, tRNA and other genes on the scaffolds are shown in gray.

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