<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

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

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

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

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

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

Whole-genome comparison maps show diversity between <i>Brevibacillus</i> phage clusters.

<p>A Phamerator [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156838#pone.0156838.ref030" target="_blank">30</a>] whole genome comparison map was prepared using a representative member of each cluster. 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. Gene products are numbered. Colored boxes correspond to encoded proteins that belong to a particular pham (family of homologous proteins) found in any of the ten <i>Brevibacillus</i> phages, while white boxes denote an orpham (an ORF not belonging to a pham). Purple, red, or green lines between genomes illustrate regions of nucleotide similarity. Emery gps 4–5 represents a frameshift event by ribosomal slippage. Functions annotated were compiled from both BLAST and CDD searches. Abbreviations used include: MGEBNA (mannosyl-glycoprotein endo-beta-N-acetylglucosamidase); XRE (XRE family transcriptional regulator); TR (Trascriptional regulator); ssDNA binding (single-stranded DNA binding); DNA rep (DNA replication); RecU (recombinase RecU); SSp DNA meth (Site-specific DNA methylase); RNA pol sigma 24 (RNA polymerase sigma 24); MD hydrolase (Metal-dependent hydrolase); DNA pol III G/T (DNA polymerase III subunit gamma/tau); Regulator of CC (Regulator of chromosome condensation family); NALAAC (N-acetylmuramoyl-L-alanine amidase CwlA); Spo0E (Sporulation protein Spo0E OR Spo0E-like sporulation regulator family); DNA pol III (DNA polymerase III); RNA pol sigma (RNA polymerase sigma factor); FtsK (Cell division protein FtsK); DNA pol I (DNA polymerase I); NALA amidase (N-acetylmuramoyl-L-alanine amidase); XRE (XRE family transcriptional regulator); Gene reg B (Accessory gene regulator B family); DNA-directed DNA pol A (DNA-directed DNA polymerase, family A); RNA pol sigma (RNA polymerase sigma factor); V spor K, ATPase (stage V sporulation protein K, ATPase AAA); Ac-di syn (cobyrinic acid ac-diamide synthase); XerD (site specific recombinase XerD); Reg s, luxR (bacterial regulatory s, luxR family); BN DNA binding (Bacterial nucleoid DNA-binding); Exc ABC (Excinuclease ABC); CJE RusA (Crossover junction endodeoxyribonuclease RusA); Asp-tRNA amidotrans B (Aspartyl-tRNA amidotransferase subunit B); ssDNA-sp exonuclease (Single-stranded DNA-specific exonuclease).</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

Multiple sequence alignment of Osiris gp55, Osiris gp59, and related homologs from <i>B</i>. <i>laterosporus</i> phages indicates high conservation.

<p>Two groups (Osiris gp55 and Osiris gp59), based on homology of related proteins, can be seen. The <i>Brevibacillus</i> phage name is followed by the gene product number and then the amino acid sequence of that gene product. Group numbers are provided to the left of the phage names. 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>] predictions for DNA binding residues are colored. Green residues differ in a group-specific manner while red residues are conserved between both groups.</p