bioRxiv preprint doi: https://doi.org/10.1101/2020.12.14.422802; this version posted December 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Title: Comparative Genomics of Three Novel Lytic Jumbo Bacteriophages Infecting 2 Staphylococcus aureus 3 4 5 Abby M. Korna,b, Andrew E. Hillhousec,d, Lichang Sunb,e, Jason J. Gilla,b,* 6 7 8 a Department of Animal Science, Texas A&M University, Kleberg Center, Suite 133 9 2471, 474 Olsen Blvd, College Station, TX 77843, [email protected] 10 b Center for Phage Technology, Texas A&M University, 300 Olsen Blvd, College Station, 11 TX 77843, USA. [email protected] 12 c Department of Veterinary Pathobiology, College of Veterinary Medicine and 13 Biomedical Sciences, Texas A&M University, College Station, TX 77843 14 d Texas A&M Institute for Genome Sciences and Society, Texas A&M University College 15 Station, TX 77884 16 e Key Laboratory of Control Technology and Standard for Agro-product Safety and 17 Quality Ministry of Agriculture, Key Laboratory of Food Quality and Safety of Jiangsu 18 Province-State Key Laboratory Breeding Base, Institute of Food Quality and Safety, 19 Jiangsu Academy of Agricultural Sciences, Nanjing, China 20 21 *Corresponding author: [email protected] 22 23 24 Running Title: Jumbo S. aureus phages 25 26 27 Key words: Staphylococcus aureus, bacteriophage, jumbo phage, MarsHill, 28 Madawaska, Machias 29 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.14.422802; this version posted December 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 30 Abstract 31 The majority of previously described Staphylococcus aureus bacteriophages belong to 32 three major groups: P68-like Podoviridae, Twort-like or K-like Myoviridae, and a more 33 diverse group of temperate Siphoviridae. Here we present three novel S. aureus 34 “jumbo” phages: MarsHill, Madawaska, and Machias. These phages were isolated from 35 swine production environments in the United States and represent a novel clade of S. 36 aureus Myoviridae that is largely unrelated to other known S. aureus phages. The 37 average genome size for these phages is ~269 kb with each genome encoding ~263 38 predicted protein-coding genes. Phage genome organization and content is most similar 39 to known jumbo phages of Bacillus, including AR9 and vB_BpuM-BpSp. All three 40 phages possess genes encoding complete viral and non-viral RNA polymerases, 41 multiple homing endonucleases, and a retron-like reverse transcriptase. Like AR9, all of 42 these phages are presumed to have uracil-substituted DNA which interferes with DNA 43 sequencing. These phages are also able to transduce host plasmids, which is 44 significant as these phages were found circulating in swine production environments 45 and can also infect human S. aureus isolates. 46 47 Importance of work: 48 This study describes the comparative genomics of three novel S. aureus jumbo phages: 49 MarsHill, Madawaska, and Machias. These three S. aureus Myoviridae represent a new 50 class of S. aureus phage that have not been described previously. These phages have 51 presumably hypermodified DNA which inhibits sequencing by several different common 52 platforms. Therefore, not only are these phages an exciting new type of S. aureus 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.14.422802; this version posted December 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 53 phage, they also represent potential genomic diversity that has been missed due to the 54 limitations of standard sequencing techniques. The data and methods presented in this 55 study could be useful for an audience far beyond those working in S. aureus phage 56 biology. This work is original and has not been submitted for publication in any other 57 journal. 58 59 Introduction 60 Staphylococcus aureus is an opportunistic pathogen of both humans and 61 animals, and is a leading cause of bacteremia, skin, soft tissue and device-related 62 infections in humans (1). The expansion and prevalence of methicillin-resistant S. 63 aureus (MRSA) imposes a significant burden to the health care system (2). S. aureus 64 infections, particularly MRSA infections, can be difficult and costly to treat, with one 65 study reporting the median cost for treatment of a MRSA surgical site infection as 66 $92,363 (3). 67 Carriage of S. aureus in the general public in the continental US ranges from 68 26% to 32% (4). An estimated 1.3% of that S. aureus being MRSA (5). However, in 69 individuals in the US that are swine farmers, production workers or veterinarians, 70 carriage of multi-drug resistant S. aureus (MDRSA) is two to six times greater than 71 individuals in the community, or those who are not exposed to swine (6, 7). As with 72 humans, S. aureus is considered to be part of the normal bacterial flora of swine (8). 73 Certain activities on swine farms such as pressure washing and tail docking generate 74 particle sizes capable of depositing primarily in human upper airways but also the 75 primary and secondary bronchi as well as terminal bronchi and alveoli (9). Livestock- 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.14.422802; this version posted December 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 76 associated methicillin-resistant S. aureus (LA-MRSA) isolates have been found to have 77 a half-life of five days in settled barn dust with an approximate die-off of 99.9% after 66- 78 72 days (10). Therefore, a better understanding of the ecology of S. aureus in these 79 environments and mitigation of MRSA in swine production facilities would be of benefit 80 to farmers and workers for safety reasons, as MRSA isolates are able to persist and 81 possibly spread throughout the environment and workers. 82 Bacteriophages (phages) are viruses that infect bacteria and are the most 83 abundant organism on Earth with an estimated 1031 in the biosphere (11). However, 84 despite their abundance and possible utility there are still many unknowns about the 85 basic biology of most phages and their interactions with their hosts in the environment. 86 Previous investigations of S. aureus phages have described three common classes of 87 phages: small, virulent P68-like Podoviridae with genomes of ~18-20 kb, various 88 temperate Siphoviridae with genomes of ~45 kb, and large, virulent Twort-like 89 Myoviridae with genomes of ~130 kb (12, 13). There is a single report of a novel large 90 S. aureus Myoviridae that appears to be distinct from the K-like Myoviridae; this phage 91 was named S6 as reported by Uchiyama et al., in 2014 (14). While S6 was estimated to 92 have a 270 kb genome by pulse-field gel electrophoresis and contain DNA in which 93 thymine was replaced by uracil, no genomic sequence for S6 has been reported. 94 Phages that have genomes over 200 kb are classified as “jumbo” phages (15). Most 95 jumbo phages have been isolated from Gram-negative hosts, with the only known 96 jumbo phages for Gram-positive hosts infecting Bacillus spp. Jumbo phage phylogeny 97 and taxonomy are complicated by their often distant relationships to each other and to 98 other known phage types; jumbo phage genomes typically encode large numbers of 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.14.422802; this version posted December 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 99 hypothetical proteins (15).This study describes three novel S. aureus jumbo phages 100 isolated from swine environments: MarsHill, Machias and Madawaska. 101 102 Methods 103 Culture and maintenance of bacteria and phages 104 S. aureus was routinely cultured on trypticase soy broth (Bacto TSB, Difco) or 105 trypticase soy agar (TSA, TSB + 1.5% w/v Bacto agar, Difco) aerobically at 30 ºC. 106 Phages were cultured using the double-layer overlay method (16) with 4 ml of top agar 107 (10 g/L Bacto Tryptone (Difco), 10 g/L NaCl, 0.5% w/v Bacto agar, (Difco)) 108 supplemented with 5 mM each CaCl2 and MgSO4 over TSA bottom plates. Lawns were 109 inoculated with 0.1 ml of a mid-log S. aureus bacterial culture grown to an OD550 of 110 ~0.5. Phage stocks were produced by the confluent plate lysate method (17) using the 111 original phage isolation host and harvested with 4-5 ml of lambda diluent (100 mM 112 NaCl, 25 mM Tris-HCl pH 7.4, 8 mM MgSO4, 0.01% w/v gelatin). Harvested lysates 113 were centrifuged at 10,000 x g, 10 min, 4 ºC, sterilized by passage through a 0.2 µm 114 syringe filter (Millipore, Burlington, MA) and stored in the dark at 4 ºC.