Characterization of CRISPR Spacer and Protospacer Sequences in Paenibacillus larvae and Its Bacteriophages

Abstract

The bacterium Paenibacillus larvae is the causative agent of American foulbrood, the most devastating bacterial disease of honeybees. Because P. larvae is antibiotic resistant, phages that infect it are currently used as alternative treatments. However, the acquisition by P. larvae of CRISPR spacer sequences from the phages could be an obstacle to treatment efforts. We searched nine complete genomes of P. larvae strains and identified 714 CRISPR spacer sequences, of which 384 are unique. Of the four epidemiologically important P. larvae strains, three of these have fewer than 20 spacers, while one strain has over 150 spacers. Of the 384 unique spacers, 18 are found as protospacers in the genomes of 49 currently sequenced P. larvae phages. One P. larvae strain does not have any protospacers found in phages, while another has eight. Protospacer distribution in the phages is uneven, with two phages having up to four protospacers, while a third of phages have none. Some phages lack protospacers found in closely related phages due to point mutations, indicating a possible escape mechanism. This study serve a point of reference for future studies on the CRISPR-Cas system in P. larvae as well as for comparative studies of other phage-host systems.

Keywords: American foulbrood; CRISPR; Paenibacillus larvae; bacterial immunity; phages; protospacers; spacers.

Figure 1

Distribution of P. larvae phages by number of unique protospacers.

Figure 2

Distribution of the 18 protospacer sequences in P. larvae phages and P. larvae strains.

Figure 3

Sequence logo of the 10 bases upstream and the 10 bases downstream of the protospacers. The -GA(A)- sequence in the 10 bases downstream is likely the PAM sequence and is downstream of 17 of the 18 protospacers.