A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPRs) are a family of DNA direct repeats found in many prokaryotic genomes. Repeats of 21-37 bp typically show weak dyad symmetry and are separated by regularly sized, nonrepetitive spacer sequences. Four CRISPR-associated (Cas) protein families, designated Cas1 to Cas4, are strictly associated with CRISPR elements and always occur near a repeat cluster. Some spacers originate from mobile genetic elements and are thought to confer "immunity" against the elements that harbor these sequences. In the present study, we have systematically investigated uncharacterized proteins encoded in the vicinity of these CRISPRs and found many additional protein families that are strictly associated with CRISPR loci across multiple prokaryotic species. Multiple sequence alignments and hidden Markov models have been built for 45 Cas protein families. These models identify family members with high sensitivity and selectivity and classify key regulators of development, DevR and DevS, in Myxococcus xanthus as Cas proteins. These identifications show that CRISPR/cas gene regions can be quite large, with up to 20 different, tandem-arranged cas genes next to a repeat cluster or filling the region between two repeat clusters. Distinctive subsets of the collection of Cas proteins recur in phylogenetically distant species and correlate with characteristic repeat periodicity. The analyses presented here support initial proposals of mobility of these units, along with the likelihood that loci of different subtypes interact with one another as well as with host cell defensive, replicative, and regulatory systems. It is evident from this analysis that CRISPR/cas loci are larger, more complex, and more heterogeneous than previously appreciated.

Figure 1. Distribution of the Different CRISPR/Cas Subtypes across Some of the Prokaryotic Species for Which a Whole-Genome Sequence Is Available

The taxonomy of each species/strain is indicated on the left side of the figure. The CRISPR/cas loci of a number of illustrative examples for the different CRISPR subtypes are displayed on the right side of the figure. ^a E. coli K12-MG1655, O157:H7 EDL933, and O157:H7 VT2-Sakai. ^b Salmonella enterica Paratyphi ATCC9150, serovar Typhi CT18, and Ty2; Salmonella typhimurium LT2 SGSC1412. ^c Y. pestis CO92, KIM, and biovar Mediaevalis 91001; Yersinia pseudotuberculosis IP32593. ^d“p” indicates a partial cluster lacking some of the genes usually associated with this subtype, the repeats, or both. Such clusters may represent autonomous functional units, degradation from the common subtype, or cases in which the missing components are supplied by distantly located CRISPR clusters within the same genome.

Figure 2. Molecular Phylogeny of the Cas1 Protein across 54 Prokaryotic Genomes

A representative selection of Cas1 protein sequences were aligned using ClustalW, and columns with greater than 20% gaps were removed. A neighbor-joining tree was calculated in Belvu using the Storm and Sonnhammer distance correction. Trees calculated using more computationally intensive methods showed insignificant differences. ^aFrom the preliminary annotation of the Haloferax volcanii genome, currently sequenced at The Institute for Genomic Research (http://www.tigr.org/tdb/mdb/mdbinprogress.html).