Characteristics and immune functions of the endogenous CRISPR-Cas systems in myxobacteria

ABSTRACT The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) system widely occurs in prokaryotic organisms to recognize and destruct genetic invaders. Systematic collation and characterization of endogenous CRISPR-Cas systems are conducive to our understanding and potential utilization of this natural genetic machinery. In this study, we screened 39 complete and 692 incomplete genomes of myxobacteria using a combined strategy to dispose of the abridged genome information and revealed at least 19 CRISPR-Cas subtypes, which were distributed with a taxonomic difference and often lost stochastically in intraspecies strains. The cas genes in each subtype were evolutionarily clustered but deeply separated, while most of the CRISPRs were divided into four types based on the motif characteristics of repeat sequences. The spacers recorded in myxobacterial CRISPRs were in high G+C content, matching lots of phages, tiny amounts of plasmids, and, surprisingly, massive organismic genomes. We experimentally demonstrated the immune and self-target immune activities of three endogenous systems in Myxococcus xanthus DK1622 against artificial genetic invaders and revealed the microhomology-mediated end-joining mechanism for the immunity-induced DNA repair but not homology-directed repair. The panoramic view and immune activities imply potential omnipotent immune functions and applications of the endogenous CRISPR-Cas machinery. IMPORTANCE Serving as an adaptive immune system, clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) empower prokaryotes to fend off the intrusion of external genetic materials. Myxobacteria are a collective of swarming Gram-stain-negative predatory bacteria distinguished by intricate multicellular social behavior. An in-depth analysis of their intrinsic CRISPR-Cas systems is beneficial for our understanding of the survival strategies employed by host cells within their environmental niches. Moreover, the experimental findings presented in this study not only suggest the robust immune functions of CRISPR-Cas in myxobacteria but also their potential applications.

Supplementary Table 6.The target design for plasmid interference assays.The spacer-matching protospacer sequences are marked in bold and red.The targets are all designed at the 5`untranslated region before the antibiotic gene apr R , under the control of the PaphII promoter.The PAM regions before the protospacers are marked in bold and black.

Table 2 .
Traced targets for CRISPR spacers of myxobacteria.The strategy for target tracing is described in the method section, and the matched sequences with a score of more than 20 are regarded as potential targets and are counted.Targets revealed some from self or other myxobacterial genomes are shown in columns with a grey background.The repetitions of the targets matching the same spacer are removed before counting.

Table 3 .
Traced targets in endogenous plasmids of myxobacteria.The targets with  6 mismatches are listed.

Table 4 .
A comparison of the genomic targets and genome-targeting spacers in myxobacterial representatives and other representative taxa.

Table 5 .
PAM Candidates and the suggested preferred PAMs for each CRISPR type.The PAM (3 nucleotides (nt) in the 5`-flanking of the protospacer) direction is shown as 5` → 3` in the table.The high-confident targets, i.e. with less than or equal to 3 nt mismatches (fetched from the IMG/VR and GenBank-Phage databases), are utilized to extract the candidate PAMs.The PAMs with a ratio of more than 10% are regarded as the preferred PAMs (marked in blue).The preferred PAMs that have the same first two bases (labeled in red) are summed as the degenerated perfectly matched targets.