View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Digital.CSIC J Appl Genet 50(4), 2009, pp. 421–430 Original article CRISPR elements in the Thermococcales: evidence for associated horizontal gene transfer in Pyrococcus furiosus M.C. Portillo, J.M. Gonzalez Institute for Natural Resources and Agrobiology, IRNAS-CSIC, Sevilla, Spain Abstract. The presence and distribution of CRISPR (clustered regularly interspaced short palindrome repeat)el- ements in the archaeal order Thermococcales were analyzed. Four complete genome sequences from the species Pyrococcus abyssi, P. furiosus, P. horikoshii, and Thermococcus kodakaraensis were studied. A fragment of the genome of P. furiosus was flanked by CRISPR elements upstream and by a single element downstream. The composition of the gene sequences contained in this genome fragment (positions 699013 to 855319) showed sig- nificant differences from the other genes in the P. furiosus genome. Differences were observed in the GC content at the third codon positions and the frequency of codon usage between the genes located in the analyzed fragment and the other genes in the P. furiosus genome. These results represent the first evidence suggesting that repeated CRISPR elements can be involved in horizontal gene transfer and genomic differentiation of hyperthermophilic Archaea. Keywords: CRISPR, gene transfer, genome, hyperthermophilic Archaea, Pyrococcus furiosus. Introduction spacers and repetitive elements modified the phage-resistance phenotype of bacteria Genomic regions consisting of tandem repeat (Barrangou et al. 2007). The incorporation of short DNA elements, typically of 21–47 base pairs (bp) fragments of foreign DNA into the genome of in length, separated by nonrepetitive spacer se- prokaryotes at CRISPR loci has been reported quences of approximately similar length, have (Bolotin et al. 2005; Pourcel et al. 2005; Godde been identified in about half of bacterial and most and Bickerton 2006). These studies only analyze archaeal genomes (Mojica et al. 1995; Jansen et al. the origin of spacers, and the addition or deletion 2002; Godde and Bickerton 2006; Lillestol et al. of repeated elements. 2006). Recent reports suggest that these short, reg- Work by Godde and Bickerton (2006) has sug- ularly spaced repeat and spacer regions (CRISPRs gested that horizontal gene transfer acts upon = clustered regularly interspaced short palindrome CRISPR-associated (cas) genes likely involving repeats) constitute a microbial immune system the use of conjugation. This transfer of genetic ma- acting similarly to eukaryotic RNA interference, terial is carried out through phages (Pourcel et al. to target and neutralize foreign DNA from phage 2005) and megaplasmids (Godde and Bickerton and plasmid sources (Makarova et al. 2006; Sorek 2006), suggesting the potential for transferring et al. 2008). Compelling evidence in support of large fragments of DNA (i.e. >40 Kb), although this hypothesis has been recently presented the spread of antibiotic resistance genes has been (Barrangou et al. 2007), demonstrating that Strep- suggested to be limited by CRISPR elements tococcus thermophilus responded to viral preda- (Marraffini and Sontheimer 2008). A recent study tion by integrating new spacers into a CRISPR (Tyson and Banfield 2008), based on comparative system. Furthermore, addition and removal of genomics of populations of Leptospirillum, pro- Received: May 4, 2009. Accepted: July 3, 2009. Correspondence: J.M. Gonzalez, Instituto de Recursos Naturales y Agrobiologia, IRNAS-CSIC, Avda. Reina Mercedes 10, 41012 – Sevilla, Spain; e-mail: [email protected] 422 M.C. Portillo, J.M. Gonzalez poses recent lateral transfer of CRISPR loci, fol- codon position for the genes in the target DNA lowed by significant loss and gain of spacer and fragment and the whole genome. GC content at the repeated element sequences. Diversification of third codon position and cumulative differences in CRISPR systems and their role in shaping species FCU were compared using portions of the evolution require further investigation (DeBoy genomic sequence of equivalent length (in number et al. 2006). of genes) to the targeted fragment being analyzed. The presence of CRISPR systems in FCU for a codon j encoding an amino acid i was hyperthermophilic Archaea has been reported for calculated as: several species (Lillestol et al. 2006; Sorek et al. nij FCU = xx/ å 2008). Among other Archaea, CRISPRs have j ij ij j=1 been found in species of Methanocaldococcus, where n represents the number of codons encod- Sulfolobus, Pyrobaculum, and Pyrococcus. ij ing for a single amino acid i. At present, there are 4 complete genomes of species The average FCU for a genomic fragment described within the order Thermococcales, including jk containing nk gene sequences was estimated as: the genera Pyrococcus and Thermococcus: P. abyssi, nk P. furiosus, P. horikoshii, and T. kodakaraensis. This AFCUj=åFCU jk /nk study analyzed the presence and distribution of k =1 CRISPR sequences in these genomes and focused The cumulative difference in AFCUj for all the on a potential horizontal gene transfer event sug- 64 codons between the genes in genome fragment gested from newly found individual elements A with respect to the genes in another portion of identical to CRISPR elements within the genome B was computed as: 64 P. furiosus genome. =- CDAB å AAFCUj×× A FCUj B j=1 Differences between the values of AFCUj for the Materials and methods genes in a selected genomic fragment and the val- ues for the rest of the genome were statistically The genome sequences and gene annotations from compared by a G-test for goodness-of-fit accord- the members of the order Thermococcales were ing to Sokal and Rohlf (1981). Comparisons be- obtained from the microbial genomes database tween the GC content at the third codon position (National Center for Biotechnology Information; for the genes contained in 2 genomic fragments http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi). were compared by the Student t-test (Sokal and Four genomes were studied within this order: Rohlf, 1981). Pyrococcus abyssi, P. furiosus, P. horikoshii, and Dendrograms to show the distance between the Thermococcus kodakaraensis. Searches for repet- genes in the different gene classes within the itive sequences throughout these genomes were P. furiosus genome and genes in the analyzed frag- performed by the software FIRES (short for Find- ment, as well as the relationship of the genes in the ing Repetitive Sequences) written in C by us and analyzed fragment with the genes in the genomes run in a computer under the Linux operating sys- of 4 species of Thermococcales, were constructed tem. This program finds every sequence repeated using the UPGMA algorithm (Garcia-Vallvé et al. over a determined number of times and over a 2000) based on the AFCUj values for the genes con- length of a specified number of nucleotides. The tained in each genome or genome fragment. repeated sequences found in these genomes, their number of repetitions, and location, were reported in the program output. (FIRES is available online Results and discussion at http://www.microextreme.net/software). The detected repeated sequences were compared with CRISPR-type motifs have been reported in most those reported at the CRISPR database Bacteria and Archaea. They have been assigned a (http://crispr.u-psud.fr/crispr/CRISPRdatabase.php) major role in controlling viral re-infections for the 4 analyzed genomes. (Barrangou et al. 2007) and could be indicative of The possibility of transfer of genetic material previous viral insertions (Andersson and Banfield associated with repeated sequences was analyzed 2008). To our knowledge, there has been no re- following the procedure proposed by Gar- ported evidence of the potential implication of cia-Vallvé et al. (2000), based on the frequency of these repeats in the insertion of novel DNA frag- codon usage (FCU) and the GC content at the third ments in microbial genomes, with the exception of CRISPR elements in the Thermococcales 423 transfer events for cas genes (Godde and could be detected within a genome, although they Bickerton 2006; Tyson and Banfield 2008). With showed an elevated proportion of conserved nu- this perspective in mind, the presence and distribu- cleotides (Table 1). Even if distinct genomes tion of CRISPR elements and identical motifs within the Thermococcales are compared, the re- were analyzed in the Thermococcales. peated motifs are generally well conserved. The repeated elements detected in the 4 studied The detected repeated elements have been genomes from the Thermococcales are reported in compared to those previously reported (CRISPR Table 1, which represents an update from previous database), and some novel elements have been dis- estimates, mainly on the detection of a couple of covered. The presence at a couple of sites of individual elements identical to those repeated se- nonrepeated elements identical to the repeated ele- quences forming CRISPR. The repeated elements ments in CRISPR sequences was only found in the in the circular genomes are mainly distributed genome of P. furiosus (Table 1). These sequences, from near the beginning of the genome sequence identical to the repeated elements found as indi- up to 240 degrees. No CRISPR elements were de- vidual elements in P. furiosus genome, were fur- tected between 240 and 358 degrees in the studied ther investigated. Specifically, a fragment of genomes. The length of these repeated