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

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.

Diversity and Distribution of a Novel Genus of Hyperthermophilic Aquificae Viruses Encoding a Proof-Reading Family-A DNA Polymerase.

Despite the high abundance of Aquificae in many geothermal systems, these bacteria are difficult to culture and no viruses infecting members of this phylum have been isolated. Here, we describe the complete, circular dsDNA Uncultivated Virus Genome (UViG) of Thermocrinis Octopus Spring virus (TOSV), derived from metagenomic data, along with eight related UViGs representing three additional viral species. Despite low overall similarity among viruses from different hot springs, the genomes shared a high degree of synteny, and encoded numerous genes for nucleotide metabolism, including a PolA-type DNA polymerase polyprotein with likely accessory functions, a DNA Pol III sliding clamp, a thymidylate kinase, a DNA gyrase, a helicase, and a DNA methylase. Also present were conserved genes predicted to code for phage capsid, large and small subunits of terminase, portal protein, holin, and lytic transglycosylase, all consistent with a distant relatedness to cultivated Caudovirales. These viruses are predicted to infect Aquificae, as multiple CRISPR spacers matching the viral genomes were identified within the genomes and metagenomic contigs from these bacteria. Based on the predicted atypical bi-directional replication strategy, low sequence similarity to known viral genomes, and unique position in gene-sharing networks, we propose a new putative genus, "Pyrovirus," in the order Caudovirales.

Genomic Analysis of 48 Paenibacillus larvae Bacteriophages.

The antibiotic-resistant bacterium Paenibacillus larvae is the causative agent of American foulbrood (AFB), currently the most destructive bacterial disease in honeybees. Phages that infect P. larvae were isolated as early as the 1950s, but it is only in recent years that P. larvae phage genomes have been sequenced and annotated. In this study we analyze the genomes of all 48 currently sequenced P. larvae phage genomes and classify them into four clusters and a singleton. The majority of P. larvae phage genomes are in the 38⁻45 kbp range and use the cohesive ends (cos) DNA-packaging strategy, while a minority have genomes in the 50⁻55 kbp range that use the direct terminal repeat (DTR) DNA-packaging strategy. The DTR phages form a distinct cluster, while the cos phages form three clusters and a singleton. Putative functions were identified for about half of all phage proteins. Structural and assembly proteins are located at the front of the genome and tend to be conserved within clusters, whereas regulatory and replication proteins are located in the middle and rear of the genome and are not conserved, even within clusters. All P. larvae phage genomes contain a conserved N-acetylmuramoyl-l-alanine amidase that serves as an endolysin.

Complete Genome Sequences of Paenibacillus larvae Phages BN12, Dragolir, Kiel007, Leyra, Likha, Pagassa, PBL1c, and Tadhana.

We present here the complete genomes of eight phages that infect Paenibacillus larvae, the causative agent of American foulbrood in honeybees. Phage PBL1c was originally isolated in 1984 from a P. larvae lysogen, while the remaining phages were isolated in 2014 from bee debris, honeycomb, and lysogens from three states in the USA.

Complete Genome Sequences of 18 Paenibacillus larvae Phages from the Western United States.

We present here the complete genomes of 18 phages that infect Paenibacillus larvae, the causative agent of American foulbrood in honeybees. The phages were isolated between 2014 and 2016 as part of an undergraduate phage discovery course at Brigham Young University. The phages were isolated primarily from bee debris and lysogens.

Comparative genomics of 9 novel Paenibacillus larvae bacteriophages.

American Foulbrood Disease, caused by the bacterium Paenibacillus larvae, is one of the most destructive diseases of the honeybee, Apis mellifera. Our group recently published the sequences of 9 new phages with the ability to infect and lyse P. larvae. Here, we characterize the genomes of these P. larvae phages, compare them to each other and to other sequenced P. larvae phages, and putatively identify protein function. The phage genomes are 38-45 kb in size and contain 68-86 genes, most of which appear to be unique to P. larvae phages. We classify P. larvae phages into 2 main clusters and one singleton based on nucleotide sequence identity. Three of the new phages show sequence similarity to other sequenced P. larvae phages, while the remaining 6 do not. We identified functions for roughly half of the P. larvae phage proteins, including structural, assembly, host lysis, DNA replication/metabolism, regulatory, and host-related functions. Structural and assembly proteins are highly conserved among our phages and are located at the start of the genome. DNA replication/metabolism, regulatory, and host-related proteins are located in the middle and end of the genome, and are not conserved, with many of these genes found in some of our phages but not others. All nine phages code for a conserved N-acetylmuramoyl-L-alanine amidase. Comparative analysis showed the phages use the "cohesive ends with 3' overhang" DNA packaging strategy. This work is the first in-depth study of P. larvae phage genomics, and serves as a marker for future work in this area.

Complete Genome Sequences of Nine Phages Capable of Infecting Paenibacillus larvae, the Causative Agent of American Foulbrood Disease in Honeybees.

We present here the complete genome sequences of nine phages that infect Paenibacillus larvae, the causative agent of American foulbrood disease in honeybees. The phages were isolated from soil, propolis, and infected bees from three U.S. states. This is the largest number of P. larvae phage genomes sequenced in a single publication to date.