Recruitment of CRISPR-Cas systems by Tn7-like transposons

Abstract

A survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain minimal type I-F CRISPR-Cas systems that consist of fused cas8f and cas5f, cas7f, and cas6f genes and a short CRISPR array. Several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas. This minimal gene complement of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-CRISPR RNA (precrRNA) processing yielding mature crRNAs and target binding but not target cleavage that is required for interference. Phylogenetic analysis demonstrates that evolution of the CRISPR-Cas-containing transposons included a single, ancestral capture of a type I-F locus and two independent instances of type I-B loci capture. We show that the transposon-associated CRISPR arrays contain spacers homologous to plasmid and temperate phage sequences and, in some cases, chromosomal sequences adjacent to the transposon. We hypothesize that the transposon-encoded CRISPR-Cas systems generate displacement (R-loops) in the cognate DNA sites, targeting the transposon to these sites and thus facilitating their spread via plasmids and phages. These findings suggest the existence of RNA-guided transposition and fit the guns-for-hire concept whereby mobile genetic elements capture host defense systems and repurpose them for different stages in the life cycle of the element.

Keywords: CRISPR-Cas systems; Tn7 transposon; crRNA guide; target-site selection; transposition strategy.

Fig. 1.

Schematic representation of the complete and minimal type I-F CRISPR-Cas systems and Tn7 transposition. (A) Gene organization of a complete and a minimal type I-F CRISPR-Cas system lacking the genes for proteins responsible for adaptation and target cleavage. Minimal I-F systems contain fused cas8f and cas5f genes that are characteristic of this group (16). Together, these proteins can be predicted to be subunits of a minimal Cascade complex. (B) Gene structure of the Tn7 genes flanked by left (L) and right (R) end sequences. Transposition catalyzed by the TnsABC+TnsD proteins directs the transposon into a single chromosomal site (attTn7) in bacterial genomes. Transposition catalyzed by the TnsABC+TnsE proteins preferentially directs transposition into actively conjugating DNA and filamentous bacteriophage (shown by a red circle with arrows). The transposon is denoted by a rectangle in the attachment site. The DNA sequence omitted in the graphic is denoted by two slashes. See text for details.

Fig. 2.

Schematic evolutionary trees for the Cas7f, TnsA, and TnsD(TniQ) protein families. (A) The dendrogram was built using 2,905 Cas7f proteins as described in Methods (see the complete tree at ftp://ftp.ncbi.nih.gov/pub/makarova/supplement/Peters_et_al_2017). The major subtrees are collapsed and shown by triangles. The branch corresponding to the minimal I-F variant is colored in orange, and the bootstrap value for this subtree is shown. (B) The dendrogram was built using 7,023 TnsA protein sequences (see the complete tree at ftp://ftp.ncbi.nih.gov/pub/makarova/supplement/Peters_et_al_2017). The branch corresponding to TnsA in the loci containing I-F variant cas genes is colored in orange, and I-B subtype cas genes are colored in green. The CRISPR-Cas subtypes are indicated next to the respective branches. Distinct cyanobacterial strains are indicated next to the respective I-B systems. The bootstrap value for the TnsA branch associated with I-F cas genes is shown. (C) The dendrogram was built using 7,963 TnsD(TniQ) proteins (see the complete tree at ftp://ftp.ncbi.nih.gov/pub/makarova/supplement/Peters_et_al_2017). The outgroup consists of the TnsD(TniQ)-like proteins that form the sister group of those associated with the type I-F CRISPR-Cas systems but encoded by Tn6022 elements lacking CRISPR-Cas (see the complete tree for the full information). The designations are as in B.

Fig. 3.

Schematic representation of Tn7, Tn6022, and selected Tn7-like transposons containing cas genes. Genomic features recognized by the transposon-encoded TniQ protein are indicated on the left (glmS, yifB, IMPDH, yciA, and SRP-RNA). Color coding and labeling are as in Fig. 1. Elements other than Tn7 and Tn6022 are denoted by the respective TnsA tree leaves (#XX) (Tn6022 = Tree node #582) (Dataset S2). Other genes are shown in gray, and known Tn7 cargo genes are indicated. Black vertical bars indicate repeats in the element-encoded arrays. DNA sequences omitted in the graphic are indicated by two slashes. See text for details.

Fig. 4.

Phylogenetic tree of selected representatives of type I-F-associated TnsD(TniQ)-like proteins. A maximum likelihood phylogenetic tree was built as described in Methods for a selected set of TnsD(TniQ)-like proteins associated with the type I-F CRISPR-Cas variant and the same outgroup as in Fig. 2C. The numbers at internal branches indicate percent bootstrap support; only values greater than 70% are indicated. Elements located in one of the three attachment sites identified in this work are shown by color as indicated (yciA, IMPDH, and SRP-RNA); random sites are in black. The leaves of the tree for the TnsD(TniQ)-like proteins (#XX) (Dataset S2) are shown in green.

Fig. S1.

Schematic representation of the end structure of Tn7-like elements: anatomy of a Tn7 insertion. Typically, the insertion occurs at a single site about 25 bp downstream from the last codon of glmS. The Tn7 end proximal to tnsA is closest to glmS (by convention it is referred to as the “right” end). Transposition generates a target site duplication (shown in red) of the chromosomal sequence that now forms a direct repeat on either side the element. In the case of insertion of the canonical Tn7 element at the attTn7 site, this sequence is GCGGG; an 8-bp end sequence starts with TGT/CAC, and immediately after this end sequence is the first 22-bp binding site for TnsB.

Fig. 5.

Model of the two targeting pathways for Tn7 elements containing CRISPR-Cas system. Designations are as in Fig. 1.

Fig. 6.

Models of the three previously described Tn7 targeting pathways and the proposed CRISPR-Cas–facilitated transposition pathway. Representations of TnsABC+TnsD (A) and TnsABC+TnsE (B) transposition pathways, the synthetic transposition pathway that targets triplex DNA complexes with a mutant form of TnsC, TnsABC* (C), and the proposed targeting pathway mediated by Cas interference complexes (D) are shown. Known host factors that participate in the TnsD (ACP, L29) and TnsE (DnaN) pathways are also shown. See text for details and references.