Comprehensive search for accessory proteins encoded with archaeal and bacterial type III CRISPR-cas gene cassettes reveals 39 new cas gene families

Abstract

A study was undertaken to identify conserved proteins that are encoded adjacent to cas gene cassettes of Type III CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR associated) interference modules. Type III modules have been shown to target and degrade dsDNA, ssDNA and ssRNA and are frequently intertwined with cofunctional accessory genes, including genes encoding CRISPR-associated Rossman Fold (CARF) domains. Using a comparative genomics approach, and defining a Type III association score accounting for coevolution and specificity of flanking genes, we identified and classified 39 new Type III associated gene families. Most archaeal and bacterial Type III modules were seen to be flanked by several accessory genes, around half of which did not encode CARF domains and remain of unknown function. Northern blotting and interference assays in Synechocystis confirmed that one particular non-CARF accessory protein family was involved in crRNA maturation. Non-CARF accessory genes were generally diverse, encoding nuclease, helicase, protease, ATPase, transporter and transmembrane domains with some encoding no known domains. We infer that additional families of non-CARF accessory proteins remain to be found. The method employed is scalable for potential application to metagenomic data once automated pipelines for annotation of CRISPR-Cas systems have been developed. All accessory genes found in this study are presented online in a readily accessible and searchable format for researchers to audit their model organism of choice: http://accessory.crispr.dk .

Keywords: CARF; CRISPR; accessory; ancillary; archaea; auxillary; bacteria; csx1; csx3; helicase; nuclease; protease; type III.

Figure 1.

Gene maps of example Type III gene cassettes including accessory genes. Core Type III genes are drawn in red with csm/cmr numbers for Type III-A/B modules, and cas numbers for Type III-D modules. cas6 and the adaptation module genes are orange and blue respectively, also with cas gene numbers. Accessory genes are drawn in purple with the gene cluster number indicated.

Figure 2.

Gene map of the Synechocystis sp. PCC 6803 pSYSA Type III-Bv module with flanking genes. Core Type III genes are coloured red and denoted with Cmr numbers. Adaptation module genes are coloured blue and marked with Cas protein numbers. Accessory genes found in this study are coloured purple and indicated with cluster numbers. Genes deleted in mutants subject to the interference assay are marked by a dot below. None of the individual deletions resulted in a marked decrease in interference activity.

Figure 3.

Experimental investigation of accessory gene knock-out mutants in Synechocystis sp. PCC 6803. a) Interference activity of subtype III-Bv-associated accessory gene knock-out mutants. Conjugation efficiencies are calculated by the ratio of the plasmid target (pT) to the plasmid non-target (pNT, control). The conjugation efficiency of the control plasmid was set to 1 and the number of colonies for the plasmid targets was normalized to the control plasmid. Data points represent mean values and standard deviations were calculated for three independent biological replicates. The accessory gene slr7080 is included in cluster 35, slr7083 in cluster 168 and slr7088 belongs to cluster 11.‘s’, invader plasmid with protospacer in sense orientation, ‘as’, in antisense orientation, ‘c’, control plasmid without protospacer. b) Northern hybridization using a radioactively labelled transcript probe spanning CRISPR3 spacers 1–4. The knock-out mutant ∆slr7088 shows decreased CRISPR3 crRNA accumulation compared to the wildtype (WT) strain. After normalization against 5S rRNA the WT clones accumulated in average 1.24 times more CRISPR3 crRNA than the slr7088 deletion mutants. A representative of two independent experiments is shown.

Figure 4.

Three subtrees from a neighbor-joining tree of all CARF proteins found in this study. Gene (locus) ids are shown along with the subtype of the associated Type III system. The branch length corresponding to a 25 % dissimilarity at the amino acid sequence level is indicated with the ruler. Closely similar CARF proteins can associate with, and cofunction with, different subtypes of Type III systems. SisCsx1 is included in the final subtree (in bold).