Characterization of the multiple CRISPR loci on Streptomyces linear plasmid pSHK1

The complete nucleotide sequence including the novel telomere sequence of Streptomyces linear plasmid pSHK1 consists of 187,263-bp, 158 genes, in which 51 genes resemble those of the linear plasmid SCP1 of Streptomyces coelicolor A3(2), and 20 genes encode transposases. Strikingly, the repetitive CRISPRs (clustered regularly interspaced short palindromic repeats) and cas (CRISPR-associated) genes were found, including a cluster of eight cas genes, in the order cas2B-cas1B-cas3B-cas5-cas4-cas2A-cas1A-cas3A, bracketed by a pair of divergent CRISPRs, and five other dispersed CRISPRs. The cas2B-cas1B-cas3B-cas5 or cas4-cas2A-cas1A genes were co-transcribed. Protein-protein interactions between Cas5 and Cas1A, 2A, 2B, 3B were detected by yeast two-hybrids, indicating a critical role of Cas5 for the formation of protein complexes. By polymerase chain reaction and Southern hybridization, 12 cas4 genes including three on linear plasmids were found among 75 newly isolated Streptomyces strains. The paired-CRISPRs and bracketed cas were also conserved in several other Streptomyces or actinomycete species. However, unlike other bacteria, the CRISPRs-cas in pSHK1 could not provide immunity against introduction of phage Phi C31 and plasmid containing the particular spacers in Streptomyces.