Comparative Genomics of Three Novel Lytic Jumbo Bacteriophages InfectingStaphylococcus aureus

Abstract

The majority of previously described Staphylococcus aureus bacteriophages belong to three major groups: P68-like Podoviridae, Twort-like or K-like Myoviridae, and a more diverse group of temperate Siphoviridae. Here we present three novel S. aureus 'jumbo' phages: MarsHill, Madawaska, and Machias. These phages were isolated from swine production environments in the United States and represent a novel clade of S. aureus Myoviridae that is largely unrelated to other known S. aureus phages. The average genome size for these phages is ~269 kb with each genome encoding ~263 predicted protein-coding genes. Phage genome organization and content is most similar to known jumbo phages of Bacillus, including AR9 and vB_BpuM-BpSp. All three phages possess genes encoding complete viral and non-viral RNA polymerases, multiple homing endonucleases, and a retron-like reverse transcriptase. Like AR9, all of these phages are presumed to have uracil-substituted DNA which interferes with DNA sequencing. These phages are also able to transduce host plasmids, which is significant as these phages were found circulating in swine production environments and can also infect human S. aureus isolates.

Template strand deoxyuridine promoter recognition by a viral RNA polymerase

“…The fold of the AR9 nvRNAP insertion domain is different from that of the E. coli RNAP and, in fact, to any protein in the Protein Data Bank (PDB). Its sequence is also unique and found only in nvRNAPs of other jumbo phages 5,6 . Its position in the structure of the nvRNAP core is also different from that of the E. coli RNAP.…”

“…2b ). Notably, all close homologs of gp226 proteins in jumbo phages with deoxyuridine-containing genomic DNA 5,6 display high sequence conservation of the 2.1bis helix with the critical valine being absolutely conserved. This suggests that these phages employ a similar mechanism for uracil-dependent promoter recognition.…”

“… e and f , The surface electrostatic potentials of the pseudo −35 -element binding motif in the WT gp226 and A 5 gp226 mutant.…”

“… g , The ssDNA and dsDNA in vitro transcription activities of the AR9 nvRNAP WT and A 5 mutant. For each in vitro transcription experiment, two technical replicates of two biological replicates resulted in similar outcomes and one of them is shown.…”

“…Gp226 shows no sequence similarity to bacterial RNAP σ subunits which mediate the recognition of promoters in bacteria or any known transcription factor. Close orthologs of gp226 are found in the genomes of other jumbo phages that have been demonstrated or are presumed to contain uracil in their genomic DNA 5,6 .…”