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Multiple Origins and Specific Evolution of CRISPR/Cas9 Systems in Minimal Bacteria ( Mollicutes)

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Multiple Origins and Specific Evolution of CRISPR/Cas9 Systems in Minimal Bacteria ( Mollicutes)

Thomas Ipoutcha et al. Front Microbiol.

Abstract

CRISPR/Cas systems provide adaptive defense mechanisms against invading nucleic acids in prokaryotes. Because of its interest as a genetic tool, the Type II CRISPR/Cas9 system from Streptococcus pyogenes has been extensively studied. It includes the Cas9 endonuclease that is dependent on a dual-guide RNA made of a tracrRNA and a crRNA. Target recognition relies on crRNA annealing and the presence of a protospacer adjacent motif (PAM). Mollicutes are currently the bacteria with the smallest genome in which CRISPR/Cas systems have been reported. Many of them are pathogenic to humans and animals (mycoplasmas and ureaplasmas) or plants (phytoplasmas and some spiroplasmas). A global survey was conducted to identify and compare CRISPR/Cas systems found in the genome of these minimal bacteria. Complete or degraded systems classified as Type II-A and less frequently as Type II-C were found in the genome of 21 out of 52 representative mollicutes species. Phylogenetic reconstructions predicted a common origin of all CRISPR/Cas systems of mycoplasmas and at least two origins were suggested for spiroplasmas systems. Cas9 in mollicutes were structurally related to the S. aureus Cas9 except the PI domain involved in the interaction with the PAM, suggesting various PAM might be recognized by Cas9 of different mollicutes. Structure of the predicted crRNA/tracrRNA hybrids was conserved and showed typical stem-loop structures pairing the Direct Repeat part of crRNAs with the 5' region of tracrRNAs. Most mollicutes crRNA/tracrRNAs showed G + C% significantly higher than the genome, suggesting a selective pressure for maintaining stability of these secondary structures. Examples of CRISPR spacers matching with mollicutes phages were found, including the textbook case of Mycoplasma cynos strain C142 having no prophage sequence but a CRISPR/Cas system with spacers targeting prophage sequences that were found in the genome of another M. cynos strain that is devoid of a CRISPR system. Despite their small genome size, mollicutes have maintained protective means against invading DNAs, including restriction/modification and CRISPR/Cas systems. The apparent lack of CRISPR/Cas systems in several groups of species including main pathogens of humans, ruminants, and plants suggests different evolutionary routes or a lower risk of phage infection in specific ecological niches.

Keywords: CRISPR/Cas9; evolution; horizontal gene transfer; mobile genetic elements; mollicutes; mycoplasma; phage; spiroplasma.

Figures

FIGURE 1
FIGURE 1
Distribution of CRISPR systems in mollicutes. Organization of the CRISPR systems predicted in mollicutes genomes are represented on the right part. Red rectangle, cas9; red triangle, tracrRNA; pink rectangle, cas1; orange rectangle, cas2; green rectangle, csn2; gray rectangle, CDS not related to CRISPR; blue bars, CRISPR array. Above numbers indicate the number of spacers. Double bars, genome interruption; black flash, disrupted gene. The phylogenetic tree was generated using the maximum likelihood method from the concatenated multiple sequence alignments of selected 62 orthologous protein involved in translation. Main phylogenetic groups are indicated; S, spiroplasma; H, hominis; P, pneumoniae; AAP, acholeplasma/phytoplasma; M, mycoides cluster. B. subtilis was used as an outgroup. Statistical values from an Approximate Likelihood-Ratio Test are indicated on branches. Hosts are indicated by colored circles; arthropod, dark green, ruminant, pink, rodent, red, bird, blue, others mammals (cats-dogs-pigs), purple, human, black, plant/arthropod, light green, reptile, brown and fish, orange.
FIGURE 2
FIGURE 2
Phylogeny of Cas9 proteins in mollicutes and reference bacteria. Amino-acid sequences of Cas9 proteins were aligned with MSAProbs and phylogenetic tree was reconstructed with PhyML with tools available on phylogeny.fr. For M. arthritidis, A. palmae and S. syrphidicola, Cas9 protein sequences were artificially simulated from fusions of ORFs covering the disrupted gene (indicated with CONCAT). Cas9 from reference bacteria were chosen from Fonfara et al. (2014); proposed subtypes were also defined according to this work; and ∗∗ refer to the two Cas9 from S. thermophilus also described in this work. Cas9 from mollicutes are highlighted in gray. Cas9 accession numbers are available in Supplementary Table S1.
FIGURE 3
FIGURE 3
Consensus sequence of Direct Repeats of mollicutes and mycoplasmas CRISPR. DR sequences from 17 CRISPR systems of mollicutes were used to create a weblogo at http://weblogo.berkeley.edu/. These logos were somewhat similar to the one from Family F13 of DR defined in CRISPRMap. The internal motif conserved at positions 12–18 in most mycoplasmas is framed in red dots.
FIGURE 4
FIGURE 4
Predicted DR/tracrRNA hybrid secondary structure. Sequences of DR and tracrRNA were concatenated and the secondary structures of the hybrids were simulated using the mfold software at http://unafold.rna.albany. edu/. Position of the DR/tracrRNA concatenation is indicated by divergent blue arrows. Predicted stems involving DR and tracrRNA anti-repeats include a lower stem (L), a bulge (B), and an upper stem (U). For S. pyogenes, processing sites of the natural hybrid by RNAse III are indicated by red arrows. For M. gallisepticum S6, DR and tracrRNA sequences were defined based on (Chylinski et al., 2014) and our own work. N, nexus stem-loop; T, terminator. The base of the upper stem loop that is highly conserved in mycoplasmas is framed in red.
FIGURE 5
FIGURE 5
Comparative genomics of CRISPR locus in two strains of M. cynos. Syntenic CDS shared by strains C142 (MCYN0853 – MCYN0843) and 210 (MCYNC210_0918 – MCYNC210_0909) are represented by white boxes connected by dashed lines. The CRISPR/Cas9 locus found in C142 is replaced by genes encoding a Type III restriction/modification system in strain 210. The disrupted mod gene of this system is indicated by a black flash. Number of genome regions encoding prophage parts are indicated.

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