Characterization of Five Novel Brevibacillus Bacteriophages and Genomic Comparison of Brevibacillus Phages.

Brevibacillus laterosporus is a spore-forming bacterium that causes a secondary infection in beehives following European Foulbrood disease. To better understand the contributions of Brevibacillus bacteriophages to the evolution of their hosts, five novel phages (Jenst, Osiris, Powder, SecTim467, and Sundance) were isolated and characterized. When compared with the five Brevibacillus phages currently in NCBI, these phages were assigned to clusters based on whole genome and proteome synteny. Powder and Osiris, both myoviruses, were assigned to the previously described Jimmer-like cluster. SecTim467 and Jenst, both siphoviruses, formed a novel phage cluster. Sundance, a siphovirus, was assigned as a singleton phage along with the previously isolated singleton, Emery. In addition to characterizing the basic relationships between these phages, several genomic features were observed. A motif repeated throughout phages Jenst and SecTim467 was frequently upstream of genes predicted to function in DNA replication, nucleotide metabolism, and transcription, suggesting transcriptional co-regulation. In addition, paralogous gene pairs that encode a putative transcriptional regulator were identified in four Brevibacillus phages. These paralogs likely evolved to bind different DNA sequences due to variation at amino acid residues predicted to bind specific nucleotides. Finally, a putative transposable element was identified in SecTim467 and Sundance that carries genes homologous to those found in Brevibacillus chromosomes. Remnants of this transposable element were also identified in phage Jenst. These discoveries provide a greater understanding of the diversity of phages, their behavior, and their evolutionary relationships to one another and to their host. In addition, they provide a foundation with which further Brevibacillus phages can be compared

Conserved Domain search identifies several host virulence factors present in <i>Brevibacillus</i> phages.

<p>Conserved Domain search identifies several host virulence factors present in <i>Brevibacillus</i> phages.</p

<i>Brevibacillus</i> phage SecTim467 genes harboring upstream conserved motifs are primarily involved in nucleotide metabolism/replication.

<p>FIMO [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156838#pone.0156838.ref046" target="_blank">46</a>] was used to locate the conserved motif in the genome of SecTim467. This chart also shows that these motifs are located in inter-gene gaps upstream of DNA metabolism and RNA transcription genes. “N/A” indicates the gene downstream of the motif at that location had an unknown function. The motif is displayed at the bottom, with the bases of the highly conserved portions of the motif highlighted throughout the figure in red. In many cases, several genes appear immediately downstream of the conserved motif in what may be an operon. In these cases, more than one gene function is reported. Two of the discovered motif sequences are present in the reverse complement of the given coordinates and are denoted by their coordinate numbers colored in red. The word graph at the bottom of the figure is a proportional representation of instances of different nucleotides at each position in the motif that contribute to the overall consensus sequence.</p

Whole proteomic analysis supports <i>Brevibacillus</i> phage cluster assignments.

<p>Whole proteomic analysis supports <i>Brevibacillus</i> phage cluster assignments.</p

DNA binding pocket comparison of Osiris gp55, Osiris gp59, and related homologous proteins from <i>B</i>. <i>laterosporus</i> phages.

<p>DNA binding pocket comparison of Osiris gp55, Osiris gp59, and related homologous proteins from <i>B</i>. <i>laterosporus</i> phages.</p

Average Nucleotide Identity of <i>Brevibacillus laterosporus</i> phages suggests four clusters of related phages.

<p>Average Nucleotide Identity of <i>Brevibacillus laterosporus</i> phages suggests four clusters of related phages.</p

Osiris gp55 superimposed on the phage P22 c2 repressor shows similar N-terminal structure and suggests possible homodimerization prior to interaction with DNA.

<p>The predicted RaptorX [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156838#pone.0156838.ref054" target="_blank">54</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156838#pone.0156838.ref057" target="_blank">57</a>] structure for Osiris gp55 superimposed using STRAP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156838#pone.0156838.ref058" target="_blank">58</a>] on the phage P22 c2 repressor protein published by Watkins et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156838#pone.0156838.ref052" target="_blank">52</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156838#pone.0156838.ref085" target="_blank">85</a>] reveals high structural homology. The DNA binding domain as predicted by RaptorX [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156838#pone.0156838.ref054" target="_blank">54</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156838#pone.0156838.ref057" target="_blank">57</a>] in Osiris gp55 is near the N-terminus, similar to the c2 repressor in phage P22. RaptorX was unable to accurately predict tertiary structure near the gp55 C-terminus.</p

Whole-genome nucleotide dotplot analysis of <i>Brevibacillus</i> phages reveals four distinct clusters.

<p>Gepard [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156838#pone.0156838.ref023" target="_blank">23</a>] nucleotide dotplot reveals four distinct phage clusters: the Jimmer-like cluster, the Jenst-like cluster, and the Emery and Sundance singletons. Diagonal lines indicate genome similarity. Vertical and horizontal lines were added to indicate phage genome boundaries and clusters of related phages are highlighted in different colors. Single dots present are a result of smaller DNA fragments aligning with that of other phages and do not represent sufficient similarity to elicit placing a phage into a particular cluster. Analysis window with the word size of 10 base pairs was used.</p

Terminase phylogenetic analysis supports four clusters of <i>Brevibacillus</i> phages with close relationships to phages from other hosts.

<p>Terminase phylogeny for <i>Brevibacillus</i> phages alongside other similar phage terminases based on at least 25% amino acid sequence shared identity. No outgroup was used as phage terminases are highly susceptible to horizontal gene transfer. Scale represents 0.1 amino acid substitutions per site. The phylogenetic tree was aligned using MUSCLE [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156838#pone.0156838.ref067" target="_blank">67</a>] and prepared using neighbor-joining trees in MEGA6 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156838#pone.0156838.ref043" target="_blank">43</a>] with bootstrapping set to 500. Any branches with a bootstrapping value less than 50% were collapsed. Clusters of related phages are highlighted in different colors: the Jimmer-like cluster in green, the Jenst-like cluster in red, and the Emery and Sundance singletons in yellow and blue, respectively.</p

A Putative transposable region found in <i>Brevibacillus</i> phages shares homology with <i>B</i>. <i>laterosporus</i>.

<p>(A) Phamerator [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156838#pone.0156838.ref030" target="_blank">30</a>] genome map comparing transposable regions of Jenst (top), SecTim467 (middle), and Sundance (bottom). Colored boxes correspond to genes that belong to a particular pham (family of homologous proteins), while white boxes denote an orpham (an ORF not belonging to a pham). Purple, red, or green shading illustrates regions of nucleotide similarity. Boxes on top of the genome ruler indicate genes that are expressed in the forward direction while those below the ruler are expressed in the reverse direction. Red dots indicate location of an inverted repeat. Ruler numbers are in kb (1,000 bases). (B) Gepard [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156838#pone.0156838.ref023" target="_blank">23</a>] dotplot showing alignment of the putative transposable region of SecTim467 with the sequence of the <i>B</i>. <i>laterosporus</i> genome portion where nucleotide homology is present. Numbers show relative nucleotide positions on the genomes. A red dot indicates the location of an inverted repeat.</p