Natural diversity of CRISPR spacers ofThermus: evidence of local spacer acquisition and global spacer exchange

Abstract

We investigated the diversity of CRISPR spacers of Thermus communities from two locations in Italy, two in Chile and one location in Russia. Among the five sampling sites, a total of more than 7200 unique spacers belonging to different CRISPR-Cas systems types and subtypes were identified. Most of these spacers are not found in CRISPR arrays of sequenced Thermus strains. Comparison of spacer sets revealed that samples within the same area (separated by few to hundreds of metres) have similar spacer sets, which appear to be largely stable at least over the course of several years. While at further distances (hundreds of kilometres and more) the similarity of spacer sets is decreased, there are still multiple common spacers in Thermus communities from different continents. The common spacers can be reconstructed in identical or similar CRISPR arrays, excluding their independent appearance and suggesting an extensive migration of thermophilic bacteria over long distances. Several new Thermus phages were isolated in the sampling sites. Mapping of spacers to bacteriophage sequences revealed examples of local acquisition of spacers from some phages and distinct patterns of targeting of phage genomes by different CRISPR-Cas systems. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.

Spacer acquisition by Type III CRISPR–Cas system during bacteriophage infection of Thermus thermophilus

“…Amplicons corresponding to expanded CRISPR-2 and CRISPR-11 arrays were purified and subjected to high-throughput sequencing. Newly acquired spacers varied in length from 35 to 42 bp ( Supplementary Figure S3A ), which matches the naturally observed variation of Type III array spacers lengths in environmental Thermus communities ( 44 ) and lengths of spacers in T. thermophilus HB27c Type III CRISPR arrays ( Supplementary Figure S3A ). Spacer sequences were mapped on the chromosome, the megaplasmid, and the phiFa genome.…”

“…The HB27с genome ( 42 , 43 ) consists of a circular chromosome and a megaplasmid and, based on its pedigree, should encode I-B, I-C, III-A and III-B subtype CRISPR–Cas systems and ten CRISPR arrays with three different types of repeats (Figure 1A , Supplementary Table S2 ). Two arrays are assigned to the subtype I-B system, two - to subtype I-C, and the remaining six are shared by the III-A and III-B subtypes ( 44 ). Given the reported variability of T. thermophilus strains ( 68 ), we determined the full genomic sequence of the isolate maintained in our laboratory.…”

“…The HB27c culture was infected with lytic bacteriophage phiFa ( 44 ) and expansion of each of the eleven CRISPR arrays was monitored by PCR over the course of several days. Experiment was performed at low initial multiplicity of infection (MOI) since high MOI was reported to promote the appearance of resistant clones that arise due to defects in phage absorption ( 69 ).…”

“…Bacteriophages phiFa (MH673672.2) and phiKo (MH673671.3) were isolated in our laboratory ( 44 ). To prepare phage lysates, 2 ml of fresh T. thermophilus HB27c cell culture (OD 600 ∼ 0.2) was infected with a single phiFa or phiKo plaque and cultivated for 2–3 h at standard T. thermophilus growth conditions (above).…”

The ecology and evolution of microbial CRISPR-Cas adaptive immune systems

“…In addition to this theoretical approach, several papers in this issue apply comparative population studies to describe spacer diversity in natural environments, and to identify the factors that drive this diversity. Lopatina et al [91] examine the CRISPR loci of populations of the bacterium Thermus in different geographical locations in Chile, Italy and Russia. They demonstrate that within a single population of the bacterium Thermus, more spacers are shared among strains than among distant populations.…”