Comparative genomics of 9 novelPaenibacillus larvaebacteriophages

Abstract

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 Paenibacillus larvae Phages BN12, Dragolir, Kiel007, Leyra, Likha, Pagassa, PBL1c, and Tadhana

“…Genome length is in the 37 kb to 42 kb range, and the G+C content was in the 41 to 44% range, consistent with 3′ cohesive ends for P. larvae phages ( 11 ). Preliminary analysis shows that phages Pagassa and Tadhana are closely related to each other, with the other phages slightly more distant; phage Dragolir was shown to be an outlier.…”

Genomic Analysis of 48 Paenibacillus larvae Bacteriophages

“…Rather than an alphanumeric scheme for naming clusters and subclusters, as used previously [ 27 ], we named clusters and subclusters after their representative phage, which will allow for easy expansion of clusters should the number of clusters increase to more than 26. By far the largest cluster is the first cluster on the left in Figure 3 , named after phage Fern.…”

“…These are genome length, GC content, DNA packaging strategy, number of genes, gene density (genes per 1 kbp), coding fraction, and cluster they belong to. For the UNLV phages Diane, Fern, Harrison, Hayley, Paisley, Vadim, Vegas, Willow and Xenia, the number of genes differs from previous work [ 18 , 27 ] because the genome annotation of these phages was revised with the updated protocol used to annotate the more recently published phages. This also applies to phages phiIBB_Pl23, HB10c2, Diva, Rani, Redbud, Shelly, Sitara, Lily and Tripp [ 14 , 15 , 16 , 17 ], which were annotated and published by their respective groups, but were re-annotated by our group using our annotation protocol for the purposes of this paper for consistency.…”

“…Electron micrographs of several P. larvae phages are shown in Figure 12 . Additional micrographs of P. larvae phages have been published [ 10 , 15 , 20 , 27 ]. All known P. larvae phages are of the Siphoviridae morphotype with long, filamentous, non-contractile tails approximately 150 nm in length.…”

“…The first P. larvae phage to have its genome sequenced was phiIBB_Pl23, isolated in Portugal, in 2013 [ 14 ], followed by phage HB10c2 in Germany [ 15 ], and phages Diva, Lily, Rani, Redbud, Shelly, Sitara and Tripp, isolated in North Carolina [ 16 , 17 ]. In 2015, the genomes of 9 P. larvae phages from the University of Nevada, Las Vegas (UNLV) were published [ 18 ], followed by the first comparative genomics analysis of P. larvae phages [ 27 ]. Concurrently, a large number of P. larvae phages were isolated at Brigham Young University (BYU) in Utah as part of BYU’s Phage Hunters course over the period 2014–2106, and the genomes of 26 of these were published in 2018 [ 19 , 20 ].…”