Bacteriophage Annapurna is a siphovirus discovered within a soil sample collected in North Georgia. It was isolated and amplified using the host Microbacterium foliorum prior to genome sequencing. Annapurna contains 84 predicted protein-coding genes encoded across a genome 56,247 base pairs in length. Based on gene content, Annapurna is assigned to actinobacteriophage cluster EF. Like other phages in the EF cluster, Annapurna appears to lack both integrase and repressor genes and is therefore predicted to be a virulent phage.
","acknowledgements":"We would like to recognize the work of Dr. Tagide deCarvalho whose TEM images of Annapurna are included in this article, as well as the University of Pittsburgh, whose precise sequencing of Annapurna made genomic annotation possible. Lastly, we would like to thank the HHMI sponsored SEA-PHAGES program for their investment and support of undergraduate student research.
","authors":[{"affiliations":[],"departments":[""],"credit":[""],"email":null,"firstName":"First Name\tLast Name\tAffiliation\tDepartment\tORCiD\tEmail\tCredit Contributions\tAcademic Status\tCorresponding Author\tSubmitting Author\tEqual Contribution Ashtyn \tStock\tLee University\tNatural Sciences\t\tastock01@leeu.edu\tInvestigation, Writing - review & editing\tUndergraduate\t\t\tyes Sarah \tPritchard\tLee University\tNatural Sciences\t\tspritc01@leeu.edu\tInvestigation, Writing - original draft\tUndergraduate\t\t\tyes Shelby\tStone\tLee University\tNatural Sciences\t\tsstone13@leeu.edu\tInvestigation, Writing - original draft\tUndergraduate\t\t\tyes Kinley\tShelton\tLee University\tNatural Sciences\t\tkshelt02@leeu.edu\tInvestigation, Writing - original draft\tUndergraduate\t\t\tyes Emme\tClowdus\tLee University\tNatural Sciences\t\teclowd00@leeu.edu\tInvestigation, Writing - original draft\tUndergraduate\t\t\tyes Emily\tDoughtery\tLee University\tNatural Sciences\t\tedough00@leeu.edu\tInvestigation, Writing - original draft\tUndergraduate\t\t\tyes Sallie \tHicks\tLee University\tNatural Sciences\t\tshicks04@leeu.edu\tInvestigation, Writing - original draft\tUndergraduate\t\t\tyes Annabelle \tMcDaniel\tLee University\tNatural Sciences\t\tamcdan03@leeu.edu\tInvestigation, Writing - original draft\tUndergraduate\t\t\tyes Kelsey\tTowle\tLee University\tNatural Sciences\t\tktowle01@leeu.edu\tInvestigation, Writing - original draft\tUndergraduate\t\t\tyes Hannah\tJoseph\tLee University\tNatural Sciences\t\thjosep00@leeu.edu\tInvestigation, Writing - original draft\tUndergraduate\t\t\tyes Kati \tPourfarzib\tLee University\tNatural Sciences\t\tkpourf00@leeu.edu\tInvestigation, Writing - original draft\tUndergraduate\t\t\tyes Michael\tBaker\tLee University\tNatural Sciences\t\tmbaker09@leeu.edu\tInvestigation, Writing - original draft\tUndergraduate\t\t\tyes William\tMyers\tLee University\tNatural Sciences\t\twmyers01@leeu.edu\tInvestigation, Writing - original draft\tUndergraduate\t\t\tyes Jadin \tAllen\tLee University\tNatural Sciences\t\tjallen25@leeu.edu\tInvestigation, Writing - original draft\tUndergraduate\t\t\tyes Sydney \tKruger\tLee University\tNatural Sciences\t\tskruge00@leeu.edu\tInvestigation, Writing - original draft\tUndergraduate\t\t\tyes Kevin \tPerez\tLee University\tNatural Sciences\t\tkperez04@leeu.edu\tInvestigation, Writing - original draft\tUndergraduate\t\t\tyes Jessie\tHolsombeck\tLee University\tNatural Sciences\t\tjholso00@leeu.edu\tValidation, Writing - review & editing\tUndergraduate\t\t\tyes Lori\tWest\tLee University\tNatural Sciences\t\tlwest@leeuniversity.edu\tValidation, Writing - review & editing\tPI\t\t\tyes Dana\tPerry\tLee University\tNatural Sciences\t\tdperry@leeuniversity.edu\tInvestigatio","lastName":"","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United 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","image":{"url":"https://portal.micropublication.org/uploads/7be62cdd94f6c5425c6f596a2a3370be.jpg"},"imageCaption":"A) Transmission electron micrograph of Annapurna showing a caspid diamter of 67-72nm and a 145-153nm tail (n=4), imaged at the University of Maryland. B) Plaque morphology of Annapurna on Microbacterium foliorum.
","imageTitle":"Transmission electron micrograph of Annapurna
","methods":"","reagents":"","patternDescription":"Due to the increase in antibiotic-resistant bacterial infections, there is a need for alternative treatment options. A promising avenue for treatment is the use of bacteriophages (Hatfull, 2022). In this study, Microbacterium foliorum, a common soil bacterium, was used as a host to isolate and purify a novel bacteriophage. The bacteriophage Annapurna was isolated from a dry soil sample collected from a cow pasture in Catoosa County, Georgia, USA (global positioning system [GPS] 4.93376N, 85.03636W) as previously described (Zorawick et al, 2024). Briefly, the soil sample was washed with peptone-yeast extract-calcium (PYCa) liquid media and the wash then filtered through a 0.22-µm filter. The filtrate was then plated in top agar with M. foliorum and plates incubated at 30˚C for 48 hours. A clear circular plaque, with a diameter of approximately 1mm, was picked and designated Annapurna (Figure 1). Multiple rounds of plating were performed to purify Annapurna, after which a lysate was prepared and used for imaging and DNA extraction. Negative-stain (1% uranyl acetate) transmission electron microscopy (TEM) showed Annapurna to have a siphovirus morphology. (Figure 1).
Annapurna’s DNA was extracted using the Promega Wizard DNA cleanup kit, prepared for sequencing using the NEB Ultra II FS kit, and sequenced at the University of Pittsburgh using Illumina NextSeq 1000 (XLEAP-P1 kit). The resulting 100-base reads were trimmed with cutadapt 4.7 (using the option: –nextseq-trim 30) (Martin, 2011) and filtered with skewer 0.2.2 (using the options: -q 20 -Q 30 -n -l 50) prior to assembly with Newbler v2.9 (Margulies et al., 2005) and assembly with 3,761-fold coverage checked for completeness with Consed v29 (Gordon et al., 1998). Annapurna's genome is 56,247 base pairs in length and has 63.5% GC content with circularly permuted ends. Based on gene content similarity equal to or greater than 35% to phages in the Actinobacteriophage database, PhagesDB, Annapurna is assigned to the EF cluster (Pope et al., 2017; Russell and Hatfull, 2017).
The genome of Annapurna was annotated utilizing Glimmer and GeneMark (Besemer and Borodovsky, 2005; Delcher, et al., 2007) to identify potential gene coding regions. These predictions were reviewed and refined in DNA Master before functional predictions using amino acid sequences were assigned using HHpred, using the PDB_mmCIF70, Pfam-v.37., NCBI Conserved Domains, and Uniprot SwissProt Viral70 databases (Söding et al., 2005; Pope and Jacobs-Sera, 2018). BLAST, using the Actinobacteriophage and NCBI non-redundant database, was used to confirm possible functions based on similarity with known sequences (Altschul et al., 1990). PhagesDB and Phamerator, using Actino_draft database v578, compared Annapurna’s genome with other phages in the EF cluster (Cresawn, 2011; Russell and Hatfull, 2016). DeepTMHMM was used to identify transmembrane proteins while Aragon and tRNAscan-SE were used to identify tRNA sequences (Lowe and Eddy, 1997; Laslett and Canback, 2004). Default settings were used for all software during genome annotation. The annotation process revealed 84 predicted protein-coding genes, no tRNAs, and no tmRNAs. Of the protein-coding genes, 26 were assigned known putative functions while 7 could only be identified as potential membrane proteins. The remaining genes code for proteins with no known function.
As with all 42 other annotated EF phages to-date, all predicted genes in Annapurna are transcribed unidirectionally, there are two genes encoding for DnaE-like DNA polymerase alpha subunits, and there are no integrase and repressor genes can be identified, suggesting EF phages are unable to establish lysogeny (Jacobs-Sera, et al., 2020).
Nucleotide sequence accession numbers
Annapurna is available at GenBank with Accession No. PV915857 and Sequence Read Archive (SRA) No. SRX29714288.
","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403-410.
","pubmedId":"","doi":"10.1016/S0022-2836(05)80360-2"},{"reference":"Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Research 33: W451-W454.
","pubmedId":"","doi":"10.1093/nar/gki487"},{"reference":"Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics 12: 10.1186/1471-2105-12-395.
","pubmedId":"","doi":"10.1186/1471-2105-12-395"},{"reference":"Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23: 673-679.
","pubmedId":"","doi":"10.1093/bioinformatics/btm009"},{"reference":"Gordon D, Abajian C, Green P. 1998. Consed: A Graphical Tool for Sequence Finishing. Genome Research 8: 195-202.
","pubmedId":"","doi":"10.1101/gr.8.3.195"},{"reference":"Hatfull GF. 2022. Mycobacteriophages: From Petri dish to patient. PLOS Pathogens 18: e1010602.
","pubmedId":"","doi":"10.1371/journal.ppat.1010602"},{"reference":"Jacobs-Sera D, Abad LA, Alvey RM, Anders KR, Aull HG, Bhalla SS, et al., Hatfull. 2020. Genomic diversity of bacteriophages infecting Microbacterium spp. PLOS ONE 15: e0234636.
","pubmedId":"","doi":"10.1371/journal.pone.0234636"},{"reference":"Laslett D. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Research 32: 11-16.
","pubmedId":"","doi":"10.1093/nar/gkh152"},{"reference":"Lowe TM, Eddy SR. 1997. tRNAscan-SE: A Program for Improved Detection of Transfer RNA Genes in Genomic Sequence. Nucleic Acids Research 25: 955-964.
","pubmedId":"","doi":"10.1093/nar/25.5.955"},{"reference":"Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, et al., Rothberg. 2005. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437: 376-380.
","pubmedId":"","doi":"10.1038/nature03959"},{"reference":"Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17: 10.
","pubmedId":"","doi":"10.14806/ej.17.1.200 "},{"reference":"Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Hatfull. 2017. Bacteriophages of\n Gordonia\n spp. Display a Spectrum of Diversity and Genetic Relationships. mBio 8: 10.1128/mbio.01069-17.
","pubmedId":"","doi":"10.1128/mbio.01069-17"},{"reference":"Pope WH, Jacobs-Sera D. 2017. Annotation of Bacteriophage Genome Sequences Using DNA Master: An Overview. Methods in Molecular Biology,Bacteriophages : 217-229.
","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_16"},{"reference":"Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"reference":"Soding J, Biegert A, Lupas AN. 2005. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Research 33: W244-W248.
","pubmedId":"","doi":"10.1093/nar/gki408"},{"reference":"Zorawik M, Jacobs-Sera D, Freise AC, SEA-PHAGES, Reddi K. 2024. Isolation of Bacteriophages on Actinobacteria Hosts. Methods in Molecular Biology,Phage Engineering and Analysis : 273-298.
","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"title":"Genome Analysis of Microbacterium foliorum Bacteriophage Annapurna
","reviews":[],"curatorReviews":[]},{"id":"c60128ff-0082-472d-bc95-c2c226aa0650","decision":"revise","abstract":"Bacteriophage Annapurna is a siphovirus discovered within a soil sample collected in North Georgia. It was isolated and amplified using the host Microbacterium foliorum prior to genome sequencing. Annapurna contains 84 predicted protein-coding genes encoded across a genome 56,247 base pairs in length. Based on gene content, Annapurna is assigned to actinobacteriophage cluster EF. Like other phages in the EF cluster, Annapurna appears to lack both integrase and repressor genes and is therefore predicted to be a virulent phage.
","acknowledgements":"We would like to recognize the work of Dr. Tagide deCarvalho whose TEM images of Annapurna are included in this article, as well as the University of Pittsburgh, whose precise sequencing of Annapurna made genomic annotation possible. Lastly, we would like to thank the HHMI sponsored SEA-PHAGES program for their investment and support of undergraduate student research.
","authors":[{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_reviewEditing"],"email":"astock01@leeu.edu","firstName":"Ashtyn ","lastName":"Stock","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"spritc01@leeu.edu","firstName":"Sarah","lastName":"Pritchard","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"sstone13@leeu.edu","firstName":"Shelby","lastName":"Stone","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"kshelt02@leeu.edu","firstName":"Kinley","lastName":"Shelton","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["writing_originalDraft"],"email":"eclowd00@leeu.edu","firstName":"Emme","lastName":"Clowdus","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural 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by the Lee University Department of Natural Sciences.
","image":{"url":"https://portal.micropublication.org/uploads/7be62cdd94f6c5425c6f596a2a3370be.jpg"},"imageCaption":"A) Transmission electron micrograph of Annapurna showing a caspid diamter of 67-72nm and a 145-153nm tail (n=4), imaged at the University of Maryland. B) Plaque morphology of Annapurna on Microbacterium foliorum.
","imageTitle":"Transmission electron micrograph of Annapurna
","methods":"","reagents":"","patternDescription":"Due to the increase in antibiotic-resistant bacterial infections, there is a need for alternative treatment options. A promising avenue for treatment is the use of bacteriophages (Hatfull, 2022). In this study, Microbacterium foliorum, a common soil bacterium, was used as a host to isolate and purify a novel bacteriophage. The bacteriophage Annapurna was isolated from a dry soil sample collected from a cow pasture in Catoosa County, Georgia, USA (global positioning system [GPS] 4.93376N, 85.03636W) as previously described (Zorawick et al, 2024). Briefly, the soil sample was washed with peptone-yeast extract-calcium (PYCa) liquid media and the wash then filtered through a 0.22-µm filter. The filtrate was then plated in top agar with M. foliorum and plates incubated at 30˚C for 48 hours. A clear circular plaque, with a diameter of approximately 1mm, was picked and designated Annapurna (Figure 1). Multiple rounds of plating were performed to purify Annapurna, after which a lysate was prepared and used for imaging and DNA extraction. Negative-stain (1% uranyl acetate) transmission electron microscopy (TEM) showed Annapurna to have a siphovirus morphology. (Figure 1).
Annapurna’s DNA was extracted using the Promega Wizard DNA cleanup kit, prepared for sequencing using the NEB Ultra II FS kit, and sequenced at the University of Pittsburgh using Illumina NextSeq 1000 (XLEAP-P1 kit). The resulting 100-base reads were trimmed with cutadapt 4.7 (using the option: –nextseq-trim 30) (Martin, 2011) and filtered with skewer 0.2.2 (using the options: -q 20 -Q 30 -n -l 50) prior to assembly with Newbler v2.9 (Margulies et al., 2005) and assembly with 3,761-fold coverage checked for completeness with Consed v29 (Gordon et al., 1998). Annapurna's genome is 56,247 base pairs in length and has 63.5% GC content with circularly permuted ends. Based on gene content similarity equal to or greater than 35% to phages in the Actinobacteriophage database, PhagesDB, Annapurna is assigned to the EF cluster (Pope et al., 2017; Russell and Hatfull, 2017).
The genome of Annapurna was annotated utilizing Glimmer and GeneMark (Besemer and Borodovsky, 2005; Delcher, et al., 2007) to identify potential gene coding regions. These predictions were reviewed and refined in DNA Master before functional predictions using amino acid sequences were assigned using HHpred, using the PDB_mmCIF70, Pfam-v.37., NCBI Conserved Domains, and Uniprot SwissProt Viral70 databases (Söding et al., 2005; Pope and Jacobs-Sera, 2018). BLAST, using the Actinobacteriophage and NCBI non-redundant database, was used to confirm possible functions based on similarity with known sequences (Altschul et al., 1990). PhagesDB and Phamerator, using Actino_draft database v578, compared Annapurna’s genome with other phages in the EF cluster (Cresawn, 2011; Russell and Hatfull, 2016). DeepTMHMM was used to identify transmembrane proteins while Aragon and tRNAscan-SE were used to identify tRNA sequences (Lowe and Eddy, 1997; Laslett and Canback, 2004). Default settings were used for all software during genome annotation. The annotation process revealed 84 predicted protein-coding genes, no tRNAs, and no tmRNAs. Of the protein-coding genes, 26 were assigned known putative functions while 7 could only be identified as potential membrane proteins. The remaining genes code for proteins with no known function.
As with all 42 other annotated EF phages to-date, all predicted genes in Annapurna are transcribed unidirectionally, there are two genes encoding for DnaE-like DNA polymerase alpha subunits, and there are no integrase and repressor genes can be identified, suggesting EF phages are unable to establish lysogeny (Jacobs-Sera, et al., 2020).
Nucleotide sequence accession numbers
Annapurna is available at GenBank with Accession No. PV915857 and Sequence Read Archive (SRA) No. SRX29714288.
","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403-410.
","pubmedId":"","doi":"10.1016/S0022-2836(05)80360-2"},{"reference":"Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Research 33: W451-W454.
","pubmedId":"","doi":"10.1093/nar/gki487"},{"reference":"Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics 12: 10.1186/1471-2105-12-395.
","pubmedId":"","doi":"10.1186/1471-2105-12-395"},{"reference":"Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23: 673-679.
","pubmedId":"","doi":"10.1093/bioinformatics/btm009"},{"reference":"Gordon D, Abajian C, Green P. 1998. Consed: A Graphical Tool for Sequence Finishing. Genome Research 8: 195-202.
","pubmedId":"","doi":"10.1101/gr.8.3.195"},{"reference":"Hatfull GF. 2022. Mycobacteriophages: From Petri dish to patient. PLOS Pathogens 18: e1010602.
","pubmedId":"","doi":"10.1371/journal.ppat.1010602"},{"reference":"Jacobs-Sera D, Abad LA, Alvey RM, Anders KR, Aull HG, Bhalla SS, et al., Hatfull. 2020. Genomic diversity of bacteriophages infecting Microbacterium spp. PLOS ONE 15: e0234636.
","pubmedId":"","doi":"10.1371/journal.pone.0234636"},{"reference":"Laslett D. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Research 32: 11-16.
","pubmedId":"","doi":"10.1093/nar/gkh152"},{"reference":"Lowe TM, Eddy SR. 1997. tRNAscan-SE: A Program for Improved Detection of Transfer RNA Genes in Genomic Sequence. Nucleic Acids Research 25: 955-964.
","pubmedId":"","doi":"10.1093/nar/25.5.955"},{"reference":"Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, et al., Rothberg. 2005. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437: 376-380.
","pubmedId":"","doi":"10.1038/nature03959"},{"reference":"Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17: 10.
","pubmedId":"","doi":"10.14806/ej.17.1.200 "},{"reference":"Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Hatfull. 2017. Bacteriophages of\n Gordonia\n spp. Display a Spectrum of Diversity and Genetic Relationships. mBio 8: 10.1128/mbio.01069-17.
","pubmedId":"","doi":"10.1128/mbio.01069-17"},{"reference":"Pope WH, Jacobs-Sera D. 2017. Annotation of Bacteriophage Genome Sequences Using DNA Master: An Overview. Methods in Molecular Biology,Bacteriophages : 217-229.
","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_16"},{"reference":"Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"reference":"Soding J, Biegert A, Lupas AN. 2005. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Research 33: W244-W248.
","pubmedId":"","doi":"10.1093/nar/gki408"},{"reference":"Zorawik M, Jacobs-Sera D, Freise AC, SEA-PHAGES, Reddi K. 2024. Isolation of Bacteriophages on Actinobacteria Hosts. Methods in Molecular Biology,Phage Engineering and Analysis : 273-298.
","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"title":"Genome Analysis of Microbacterium foliorum Bacteriophage Annapurna
","reviews":[{"reviewer":{"displayName":"Maria Diane Gainey Dr."},"openAcknowledgement":true,"status":{"submitted":true}},{"reviewer":{"displayName":"Adam D Rudner"},"openAcknowledgement":null,"status":{"submitted":false}},{"reviewer":{"displayName":"Dustin Edwards"},"openAcknowledgement":true,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"5148de46-fa02-40f8-b3f7-d2d94669aa2b","decision":"accept","abstract":"Bacteriophage Annapurna is a siphovirus discovered within a soil sample collected in North Georgia. Annapurna was isolated and amplified using the host Microbacterium foliorum prior to genome sequencing. Annapurna contains 84 predicted protein-coding genes encoded across a genome 56,247 base pairs in length. Based on gene content, Annapurna is assigned to actinobacteriophage cluster EF. Like other phages in the EF cluster, Annapurna appears to lack both integrase and repressor genes and is therefore predicted to be a virulent phage.
","acknowledgements":"We would like to recognize the work of Dr. Tagide deCarvalho whose TEM images of Annapurna are included in this article, as well as the University of Pittsburgh, whose precise sequencing of Annapurna made genomic annotation possible. Lastly, we would like to thank the HHMI sponsored SEA-PHAGES program for their investment and support of undergraduate student research.
","authors":[{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_reviewEditing"],"email":"astock01@leeu.edu","firstName":"Ashtyn ","lastName":"Stock","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"spritc01@leeu.edu","firstName":"Sarah","lastName":"Pritchard","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"sstone13@leeu.edu","firstName":"Shelby","lastName":"Stone","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"kshelt02@leeu.edu","firstName":"Kinley","lastName":"Shelton","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["writing_originalDraft"],"email":"eclowd00@leeu.edu","firstName":"Emme","lastName":"Clowdus","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"edough00@leeu.edu","firstName":"Emily","lastName":"Doughtery","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"shicks04@leeu.edu","firstName":"Sallie","lastName":"Hicks","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"amcdan03@leeu.edu","firstName":"Annabelle ","lastName":"McDaniel","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"ktowle01@leeu.edu","firstName":"Kelsey","lastName":"Towle","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"hjosep00@leeu.edu","firstName":"Hannah","lastName":"Joseph","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"kpourf00@leeu.edu","firstName":"Kati","lastName":"Pourfarzib","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"mbaker09@leeu.edu","firstName":"Michael","lastName":"Baker","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"wmyers01@leeu.edu","firstName":"William","lastName":"Myers","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"jallen25@leeu.edu","firstName":"Jadin","lastName":"Allen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"skruge00@leeu.edu","firstName":"Sydney","lastName":"Kruger","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"kperez04@leeu.edu","firstName":"Kevin","lastName":"Perez","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["validation","writing_reviewEditing"],"email":"jholso00@leeu.edu","firstName":"Jessie","lastName":"Holsombeck","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["validation","writing_reviewEditing"],"email":"lwest@leeuniversity.edu","firstName":"Lori","lastName":"West","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","visualization"],"email":"dperry@leeuniversity.edu","firstName":"Dana","lastName":"Perry","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["validation","writing_reviewEditing"],"email":"jdaft@leeuniversity.edu","firstName":"Joseph","lastName":"Daft","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":null,"extendedData":[],"funding":"This project was funded by the Lee University Department of Natural Sciences.
","image":{"url":"https://portal.micropublication.org/uploads/4ce9dd7842d0250df7ca798f2150e8e9.jpg"},"imageCaption":"A) Transmission electron micrograph of Annapurna showing a caspid diamter of 67-72nm and a 145-153nm tail (n=4), imaged at the University of Maryland. B) Plaque morphology of Annapurna plated with Microbacterium foliorum.
","imageTitle":"Transmission electron micrograph of an Annapurna virion and plaque morphology
","methods":"","reagents":"","patternDescription":"Due to the increase in antibiotic-resistant bacterial infections, there is a need for alternative treatment options. A promising avenue for treatment is the use of bacteriophages (Hatfull, 2022). In this study, Microbacterium foliorum NRRL B-24224, a common soil bacterium, was used as a host to isolate and purify a novel bacteriophage. The bacteriophage Annapurna was isolated from a dry soil sample collected from a cow pasture in Catoosa County, Georgia, USA (global positioning system [GPS] 34.56015N, 85.02109W) as previously described (Zorawik et al, 2024). Briefly, the soil sample was washed with peptone-yeast extract-calcium (PYCa) liquid media and the wash then filtered through a 0.22-µm filter. The filtrate was then plated in top agar with M. foliorum and plates incubated at 30˚C for 48 hours. A clear circular plaque, with a mean diameter of 1.90mm ± 0.78mm (n=20), was picked and designated Annapurna (Figure 1). Multiple rounds of plating were performed to purify Annapurna, after which a lysate was prepared and used for imaging and DNA extraction. Negative-stain (1% uranyl acetate) transmission electron microscopy (TEM) showed Annapurna to have a siphovirus morphology. (Figure 1).
Annapurna's DNA was extracted using the Promega Wizard DNA cleanup kit, prepared for sequencing using the NEB Ultra II FS kit, and sequenced at the University of Pittsburgh using Illumina NextSeq 1000 (XLEAP-P1 kit). The resulting 100-base reads were trimmed with cutadapt 4.7 (using the option: –nextseq-trim 30) (Martin, 2011) and filtered with skewer 0.2.2 (using the options: -q 20 -Q 30 -n -l 50) prior to assembly with Newbler v2.9 (Margulies et al., 2005) and assembly with 3,761-fold coverage checked for completeness with Consed v29 (Gordon et al., 1998). Annapurna's genome is 56,247 base pairs in length and has 63.5% GC content with circularly permuted ends. Based on gene content similarity equal to or greater than 35% to phages in the Actinobacteriophage database, PhagesDB, Annapurna is assigned to the EF cluster (Pope et al., 2017; Russell and Hatfull, 2017).
The genome of Annapurna was annotated utilizing Glimmer v3.02 and GeneMark v4.28 (Besemer and Borodovsky, 2005; Delcher, et al., 2007) to identify potential gene coding regions. These predictions were reviewed and refined in DNA Master v5.23.6 before functional predictions using amino acid sequences were assigned using HHpred MPI Bioinformatics Toolkit v3.3, using the PDB_mmCIF70, Pfam-v.37., NCBI Conserved Domains v3.21, and Uniprot SwissProt Viral70 databases (Söding et al., 2005; Pope and Jacobs-Sera, 2018). BLAST, using the Actinobacteriophage and NCBI non-redundant database, was used to confirm possible functions based on similarity with known sequences (Altschul et al., 1990). PhagesDB and Phamerator, with Actino_draft database v578, were used to compare Annapurna's genome with other phages in the EF cluster (Cresawn, 2011; Russell and Hatfull, 2016). DeepTMHMM v1.0.44 was used to identify transmembrane proteins while Aragon v1.2.41 and tRNAscan-SE v2.0.6 were used to identify tRNA sequences (Lowe and Eddy, 1997; Laslett and Canback, 2004). Default settings were used for all software during genome annotation. The annotation process revealed 84 predicted protein-coding genes, no tRNAs, and no tmRNAs. Of the protein-coding genes, 26 were assigned putative functions while 7 were identified as potential membrane proteins. The remaining genes code for proteins with no known function.
As with all 42 other annotated EF phages to-date, all predicted genes in Annapurna are transcribed unidirectionally. Additionally, there are two genes encoding for DnaE-like DNA polymerase alpha subunits and no integrase and repressor genes were identified, suggesting EF phages are unable to establish lysogeny (Jacobs-Sera, et al., 2020).
Nucleotide sequence accession numbers
Annapurna is available at GenBank with Accession No. PV915857 and Sequence Read Archive (SRA) No. SRX29714288.
","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403-410.
","pubmedId":"","doi":"10.1016/S0022-2836(05)80360-2"},{"reference":"Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Research 33: W451-W454.
","pubmedId":"","doi":"10.1093/nar/gki487"},{"reference":"Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics 12: 10.1186/1471-2105-12-395.
","pubmedId":"","doi":"10.1186/1471-2105-12-395"},{"reference":"Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23: 673-679.
","pubmedId":"","doi":"10.1093/bioinformatics/btm009"},{"reference":"Gordon D, Abajian C, Green P. 1998. Consed: A Graphical Tool for Sequence Finishing. Genome Research 8: 195-202.
","pubmedId":"","doi":"10.1101/gr.8.3.195"},{"reference":"Hatfull GF. 2022. Mycobacteriophages: From Petri dish to patient. PLOS Pathogens 18: e1010602.
","pubmedId":"","doi":"10.1371/journal.ppat.1010602"},{"reference":"Jacobs-Sera D, Abad LA, Alvey RM, Anders KR, Aull HG, Bhalla SS, et al., Hatfull. 2020. Genomic diversity of bacteriophages infecting Microbacterium spp. PLOS ONE 15: e0234636.
","pubmedId":"","doi":"10.1371/journal.pone.0234636"},{"reference":"Laslett D. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Research 32: 11-16.
","pubmedId":"","doi":"10.1093/nar/gkh152"},{"reference":"Lowe TM, Eddy SR. 1997. tRNAscan-SE: A Program for Improved Detection of Transfer RNA Genes in Genomic Sequence. Nucleic Acids Research 25: 955-964.
","pubmedId":"","doi":"10.1093/nar/25.5.955"},{"reference":"Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, et al., Rothberg. 2005. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437: 376-380.
","pubmedId":"","doi":"10.1038/nature03959"},{"reference":"Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17: 10.
","pubmedId":"","doi":"10.14806/ej.17.1.200 "},{"reference":"Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Hatfull. 2017. Bacteriophages of\n Gordonia\n spp. Display a Spectrum of Diversity and Genetic Relationships. mBio 8: 10.1128/mbio.01069-17.
","pubmedId":"","doi":"10.1128/mbio.01069-17"},{"reference":"Pope WH, Jacobs-Sera D. 2017. Annotation of Bacteriophage Genome Sequences Using DNA Master: An Overview. Methods in Molecular Biology,Bacteriophages : 217-229.
","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_16"},{"reference":"Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"reference":"Soding J, Biegert A, Lupas AN. 2005. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Research 33: W244-W248.
","pubmedId":"","doi":"10.1093/nar/gki408"},{"reference":"Zorawik M, Jacobs-Sera D, Freise AC, SEA-PHAGES, Reddi K. 2024. Isolation of Bacteriophages on Actinobacteria Hosts. Methods in Molecular Biology,Phage Engineering and Analysis : 273-298.
","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"title":"Genome Analysis of Microbacterium foliorum Bacteriophage Annapurna
","reviews":[{"reviewer":{"displayName":"Dustin Edwards"},"openAcknowledgement":true,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"3a56260b-45f5-49be-90f5-d97380507be8","decision":"accept","abstract":"Bacteriophage Annapurna is a siphovirus discovered within a soil sample collected in North Georgia. Annapurna was isolated and amplified using the host Microbacterium foliorum prior to genome sequencing. Annapurna contains 84 predicted protein-coding genes encoded across a genome 56,247 base pairs in length. Based on gene content, Annapurna is assigned to actinobacteriophage cluster EF. Like other phages in the EF cluster, Annapurna appears to lack both integrase and repressor genes and is therefore predicted to be a virulent phage.
","acknowledgements":"We would like to recognize the work of Dr. Tagide deCarvalho whose TEM images of Annapurna are included in this article, as well as the University of Pittsburgh, whose precise sequencing of Annapurna made genomic annotation possible. Lastly, we would like to thank the HHMI sponsored SEA-PHAGES program for their investment and support of undergraduate student research.
","authors":[{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_reviewEditing"],"email":"astock01@leeu.edu","firstName":"Ashtyn ","lastName":"Stock","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"spritc01@leeu.edu","firstName":"Sarah","lastName":"Pritchard","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural 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Sciences"],"credit":["validation","writing_reviewEditing"],"email":"lwest@leeuniversity.edu","firstName":"Lori","lastName":"West","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","visualization"],"email":"dperry@leeuniversity.edu","firstName":"Dana","lastName":"Perry","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["validation","writing_reviewEditing"],"email":"jdaft@leeuniversity.edu","firstName":"Joseph","lastName":"Daft","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":null,"extendedData":[],"funding":"This project was funded by the Lee University Department of Natural Sciences.
","image":{"url":"https://portal.micropublication.org/uploads/4ce9dd7842d0250df7ca798f2150e8e9.jpg"},"imageCaption":"A) Transmission electron micrograph of Annapurna showing a capsid diamter of 67-72nm and a 145-153nm tail (n=4), imaged at the University of Maryland Baltimore County. B) Plaque morphology of Annapurna plated with Microbacterium foliorum.
","imageTitle":"Transmission electron micrograph of an Annapurna virion and plaque morphology
","methods":"","reagents":"","patternDescription":"Due to the increase in antibiotic-resistant bacterial infections, there is a need for alternative treatment options. A promising avenue for treatment is the use of bacteriophages (Hatfull, 2022). In this study, Microbacterium foliorum NRRL B-24224, a common soil bacterium, was used as a host to isolate and purify a novel bacteriophage. The bacteriophage Annapurna was isolated from a dry soil sample collected from a cow pasture in Catoosa County, Georgia, USA (global positioning system [GPS] 34.56015N, 85.02109W) as previously described (Zorawik et al, 2024). Briefly, the soil sample was washed with peptone-yeast extract-calcium (PYCa) liquid media and the wash then filtered through a 0.22-µm filter. The filtrate was then plated in top agar with M. foliorum and plates incubated at 30˚C for 48 hours. A clear circular plaque, with a mean diameter of 1.90mm ± 0.78mm (n=20), was picked and designated Annapurna (Figure 1). Multiple rounds of plating were performed to purify Annapurna, after which a lysate was prepared and used for imaging and DNA extraction. Negative-stain (1% uranyl acetate) transmission electron microscopy (TEM) showed Annapurna to have a siphovirus morphology. (Figure 1).
Annapurna's DNA was extracted using the Promega Wizard DNA cleanup kit, prepared for sequencing using the NEB Ultra II FS kit, and sequenced at the University of Pittsburgh using Illumina NextSeq 1000 (XLEAP-P1 kit). The resulting 100-base reads were trimmed with cutadapt 4.7 (using the option: –nextseq-trim 30) (Martin, 2011) and filtered with skewer 0.2.2 (using the options: -q 20 -Q 30 -n -l 50) prior to assembly with Newbler v2.9 (Margulies et al., 2005) and assembly with 3,761-fold coverage checked for completeness with Consed v29 (Gordon et al., 1998). Annapurna's genome is 56,247 base pairs in length and has 63.5% GC content with circularly permuted ends. Based on gene content similarity equal to or greater than 35% to phages in the Actinobacteriophage database, PhagesDB, Annapurna is assigned to the EF cluster (Pope et al., 2017; Russell and Hatfull, 2017).
The genome of Annapurna was annotated utilizing Glimmer v3.02 and GeneMark v4.28 (Besemer and Borodovsky, 2005; Delcher, et al., 2007) to identify potential gene coding regions. These predictions were reviewed and refined in DNA Master v5.23.6 before functional predictions using amino acid sequences were assigned using HHpred MPI Bioinformatics Toolkit v3.3, using the PDB_mmCIF70, Pfam-v.37., NCBI Conserved Domains v3.21, and Uniprot SwissProt Viral70 databases (Söding et al., 2005; Pope and Jacobs-Sera, 2018). BLAST, using the Actinobacteriophage and NCBI non-redundant database, was used to confirm possible functions based on similarity with known sequences (Altschul et al., 1990). PhagesDB and Phamerator, with Actino_draft database v578, were used to compare Annapurna's genome with other phages in the EF cluster (Cresawn, 2011; Russell and Hatfull, 2016). DeepTMHMM v1.0.44 was used to identify transmembrane proteins while Aragon v1.2.41 and tRNAscan-SE v2.0.6 were used to identify tRNA sequences (Lowe and Eddy, 1997; Laslett and Canback, 2004). Default settings were used for all software during genome annotation. The annotation process revealed 84 predicted protein-coding genes, no tRNAs, and no tmRNAs. Of the protein-coding genes, 26 were assigned putative functions while 7 were identified as potential membrane proteins. The remaining genes code for proteins with no known function.
As with all 42 other annotated EF phages to-date, all predicted genes in Annapurna are transcribed unidirectionally. Additionally, there are two genes encoding for DnaE-like DNA polymerase alpha subunits and no integrase and repressor genes were identified, suggesting EF phages are unable to establish lysogeny (Jacobs-Sera, et al., 2020).
Nucleotide sequence accession numbers
Annapurna is available at GenBank with Accession No. PV915857 and Sequence Read Archive (SRA) No. SRX29714288.
","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403-410.
","pubmedId":"","doi":"10.1016/S0022-2836(05)80360-2"},{"reference":"Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Research 33: W451-W454.
","pubmedId":"","doi":"10.1093/nar/gki487"},{"reference":"Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics 12: 10.1186/1471-2105-12-395.
","pubmedId":"","doi":"10.1186/1471-2105-12-395"},{"reference":"Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23: 673-679.
","pubmedId":"","doi":"10.1093/bioinformatics/btm009"},{"reference":"Gordon D, Abajian C, Green P. 1998. Consed: A Graphical Tool for Sequence Finishing. Genome Research 8: 195-202.
","pubmedId":"","doi":"10.1101/gr.8.3.195"},{"reference":"Hatfull GF. 2022. Mycobacteriophages: From Petri dish to patient. PLOS Pathogens 18: e1010602.
","pubmedId":"","doi":"10.1371/journal.ppat.1010602"},{"reference":"Jacobs-Sera D, Abad LA, Alvey RM, Anders KR, Aull HG, Bhalla SS, et al., Hatfull. 2020. Genomic diversity of bacteriophages infecting Microbacterium spp. PLOS ONE 15: e0234636.
","pubmedId":"","doi":"10.1371/journal.pone.0234636"},{"reference":"Laslett D. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Research 32: 11-16.
","pubmedId":"","doi":"10.1093/nar/gkh152"},{"reference":"Lowe TM, Eddy SR. 1997. tRNAscan-SE: A Program for Improved Detection of Transfer RNA Genes in Genomic Sequence. Nucleic Acids Research 25: 955-964.
","pubmedId":"","doi":"10.1093/nar/25.5.955"},{"reference":"Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, et al., Rothberg. 2005. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437: 376-380.
","pubmedId":"","doi":"10.1038/nature03959"},{"reference":"Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17: 10.
","pubmedId":"","doi":"10.14806/ej.17.1.200 "},{"reference":"Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Hatfull. 2017. Bacteriophages of\n Gordonia\n spp. Display a Spectrum of Diversity and Genetic Relationships. mBio 8: 10.1128/mbio.01069-17.
","pubmedId":"","doi":"10.1128/mbio.01069-17"},{"reference":"Pope WH, Jacobs-Sera D. 2017. Annotation of Bacteriophage Genome Sequences Using DNA Master: An Overview. Methods in Molecular Biology,Bacteriophages : 217-229.
","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_16"},{"reference":"Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"reference":"Soding J, Biegert A, Lupas AN. 2005. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Research 33: W244-W248.
","pubmedId":"","doi":"10.1093/nar/gki408"},{"reference":"Zorawik M, Jacobs-Sera D, Freise AC, SEA-PHAGES, Reddi K. 2024. Isolation of Bacteriophages on Actinobacteria Hosts. Methods in Molecular Biology,Phage Engineering and Analysis : 273-298.
","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"title":"Genome Analysis of Microbacterium foliorum Bacteriophage Annapurna
","reviews":[],"curatorReviews":[]},{"id":"a88d15e3-a4c9-44f8-96d3-cd17720340ac","decision":"publish","abstract":"Bacteriophage Annapurna is a siphovirus discovered within a soil sample collected in North Georgia. Annapurna was isolated and amplified using the host Microbacterium foliorum prior to genome sequencing. Annapurna contains 84 predicted protein-coding genes encoded across a genome 56,247 base pairs in length. Based on gene content, Annapurna is assigned to actinobacteriophage cluster EF. Like other phages in the EF cluster, Annapurna appears to lack both integrase and repressor genes and is therefore predicted to be a virulent phage.
","acknowledgements":"We would like to recognize the work of Dr. Tagide deCarvalho whose TEM images of Annapurna are included in this article, as well as the University of Pittsburgh, whose precise sequencing of Annapurna made genomic annotation possible. Lastly, we would like to thank the HHMI sponsored SEA-PHAGES program for their investment and support of undergraduate student research.
","authors":[{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_reviewEditing"],"email":"astock01@leeu.edu","firstName":"Ashtyn ","lastName":"Stock","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"spritc01@leeu.edu","firstName":"Sarah","lastName":"Pritchard","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"sstone13@leeu.edu","firstName":"Shelby","lastName":"Stone","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"kshelt02@leeu.edu","firstName":"Kinley","lastName":"Shelton","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["writing_originalDraft"],"email":"eclowd00@leeu.edu","firstName":"Emme","lastName":"Clowdus","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"edough00@leeu.edu","firstName":"Emily","lastName":"Doughtery","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"shicks04@leeu.edu","firstName":"Sallie","lastName":"Hicks","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"amcdan03@leeu.edu","firstName":"Annabelle ","lastName":"McDaniel","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"ktowle01@leeu.edu","firstName":"Kelsey","lastName":"Towle","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"hjosep00@leeu.edu","firstName":"Hannah","lastName":"Joseph","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"kpourf00@leeu.edu","firstName":"Kati","lastName":"Pourfarzib","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"mbaker09@leeu.edu","firstName":"Michael","lastName":"Baker","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"wmyers01@leeu.edu","firstName":"William","lastName":"Myers","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"jallen25@leeu.edu","firstName":"Jadin","lastName":"Allen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"skruge00@leeu.edu","firstName":"Sydney","lastName":"Kruger","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"kperez04@leeu.edu","firstName":"Kevin","lastName":"Perez","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["validation","writing_reviewEditing"],"email":"jholso00@leeu.edu","firstName":"Jessie","lastName":"Holsombeck","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["validation","writing_reviewEditing"],"email":"lwest@leeuniversity.edu","firstName":"Lori","lastName":"West","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["investigation","visualization"],"email":"dperry@leeuniversity.edu","firstName":"Dana","lastName":"Perry","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Lee University, Cleveland, Tennessee, United States"],"departments":["Natural Sciences"],"credit":["validation","writing_reviewEditing"],"email":"jdaft@leeuniversity.edu","firstName":"Joseph","lastName":"Daft","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This project was funded by the Lee University Department of Natural Sciences.
","image":{"url":"https://portal.micropublication.org/uploads/4ce9dd7842d0250df7ca798f2150e8e9.jpg"},"imageCaption":"A) Transmission electron micrograph of Annapurna showing a capsid diamter of 67-72nm and a 145-153nm tail (n=4), imaged at the University of Maryland Baltimore County. B) Plaque morphology of Annapurna plated with Microbacterium foliorum.
","imageTitle":"Transmission electron micrograph of an Annapurna virion and plaque morphology
","methods":"","reagents":"","patternDescription":"Due to the increase in antibiotic-resistant bacterial infections, there is a need for alternative treatment options. A promising avenue for treatment is the use of bacteriophages (Hatfull, 2022). In this study, Microbacterium foliorum NRRL B-24224, a common soil bacterium, was used as a host to isolate and purify a novel bacteriophage. The bacteriophage Annapurna was isolated from a dry soil sample collected from a cow pasture in Catoosa County, Georgia, USA (global positioning system [GPS] 34.56015N, 85.02109W) as previously described (Zorawik et al, 2024). Briefly, the soil sample was washed with peptone-yeast extract-calcium (PYCa) liquid media and the wash then filtered through a 0.22-µm filter. The filtrate was then plated in top agar with M. foliorum and plates incubated at 30˚C for 48 hours. A clear circular plaque, with a mean diameter of 1.90mm ± 0.78mm (n=20), was picked and designated Annapurna (Figure 1). Multiple rounds of plating were performed to purify Annapurna, after which a lysate was prepared and used for imaging and DNA extraction. Negative-stain (1% uranyl acetate) transmission electron microscopy (TEM) showed Annapurna to have a siphovirus morphology. (Figure 1).
Annapurna's DNA was extracted using the Promega Wizard DNA cleanup kit, prepared for sequencing using the NEB Ultra II FS kit, and sequenced at the University of Pittsburgh using Illumina NextSeq 1000 (XLEAP-P1 kit). The resulting 100-base reads were trimmed with cutadapt 4.7 (using the option: –nextseq-trim 30) (Martin, 2011) and filtered with skewer 0.2.2 (using the options: -q 20 -Q 30 -n -l 50) prior to assembly with Newbler v2.9 (Margulies et al., 2005) and assembly with 3,761-fold coverage checked for completeness with Consed v29 (Gordon et al., 1998). Annapurna's genome is 56,247 base pairs in length and has 63.5% GC content with circularly permuted ends. Based on gene content similarity equal to or greater than 35% to phages in the Actinobacteriophage database, PhagesDB, Annapurna is assigned to the EF cluster (Pope et al., 2017; Russell and Hatfull, 2017).
The genome of Annapurna was annotated utilizing Glimmer v3.02 and GeneMark v4.28 (Besemer and Borodovsky, 2005; Delcher, et al., 2007) to identify potential gene coding regions. These predictions were reviewed and refined in DNA Master v5.23.6 before functional predictions using amino acid sequences were assigned using HHpred MPI Bioinformatics Toolkit v3.3, using the PDB_mmCIF70, Pfam-v.37., NCBI Conserved Domains v3.21, and Uniprot SwissProt Viral70 databases (Söding et al., 2005; Pope and Jacobs-Sera, 2018). BLAST, using the Actinobacteriophage and NCBI non-redundant database, was used to confirm possible functions based on similarity with known sequences (Altschul et al., 1990). PhagesDB and Phamerator, with Actino_draft database v578, were used to compare Annapurna's genome with other phages in the EF cluster (Cresawn, 2011; Russell and Hatfull, 2016). DeepTMHMM v1.0.44 was used to identify transmembrane proteins while Aragon v1.2.41 and tRNAscan-SE v2.0.6 were used to identify tRNA sequences (Lowe and Eddy, 1997; Laslett and Canback, 2004). Default settings were used for all software during genome annotation. The annotation process revealed 84 predicted protein-coding genes, no tRNAs, and no tmRNAs. Of the protein-coding genes, 26 were assigned putative functions while 7 were identified as potential membrane proteins. The remaining genes code for proteins with no known function.
As with all 42 other annotated EF phages to-date, all predicted genes in Annapurna are transcribed unidirectionally. Additionally, there are two genes encoding for DnaE-like DNA polymerase alpha subunits and no integrase and repressor genes were identified, suggesting EF phages are unable to establish lysogeny (Jacobs-Sera, et al., 2020).
Nucleotide sequence accession numbers
Annapurna is available at GenBank with Accession No. PV915857 and Sequence Read Archive (SRA) No. SRX29714288.
","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403-410.
","pubmedId":"","doi":"10.1016/S0022-2836(05)80360-2"},{"reference":"Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Research 33: W451-W454.
","pubmedId":"","doi":"10.1093/nar/gki487"},{"reference":"Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics 12: 10.1186/1471-2105-12-395.
","pubmedId":"","doi":"10.1186/1471-2105-12-395"},{"reference":"Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23: 673-679.
","pubmedId":"","doi":"10.1093/bioinformatics/btm009"},{"reference":"Gordon D, Abajian C, Green P. 1998. Consed: A Graphical Tool for Sequence Finishing. Genome Research 8: 195-202.
","pubmedId":"","doi":"10.1101/gr.8.3.195"},{"reference":"Hatfull GF. 2022. Mycobacteriophages: From Petri dish to patient. PLOS Pathogens 18: e1010602.
","pubmedId":"","doi":"10.1371/journal.ppat.1010602"},{"reference":"Jacobs-Sera D, Abad LA, Alvey RM, Anders KR, Aull HG, Bhalla SS, et al., Hatfull. 2020. Genomic diversity of bacteriophages infecting Microbacterium spp. PLOS ONE 15: e0234636.
","pubmedId":"","doi":"10.1371/journal.pone.0234636"},{"reference":"Laslett D. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Research 32: 11-16.
","pubmedId":"","doi":"10.1093/nar/gkh152"},{"reference":"Lowe TM, Eddy SR. 1997. tRNAscan-SE: A Program for Improved Detection of Transfer RNA Genes in Genomic Sequence. Nucleic Acids Research 25: 955-964.
","pubmedId":"","doi":"10.1093/nar/25.5.955"},{"reference":"Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, et al., Rothberg. 2005. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437: 376-380.
","pubmedId":"","doi":"10.1038/nature03959"},{"reference":"Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17: 10.
","pubmedId":"","doi":"10.14806/ej.17.1.200 "},{"reference":"Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Hatfull. 2017. Bacteriophages of\n Gordonia\n spp. Display a Spectrum of Diversity and Genetic Relationships. mBio 8: 10.1128/mbio.01069-17.
","pubmedId":"","doi":"10.1128/mbio.01069-17"},{"reference":"Pope WH, Jacobs-Sera D. 2017. Annotation of Bacteriophage Genome Sequences Using DNA Master: An Overview. Methods in Molecular Biology,Bacteriophages : 217-229.
","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_16"},{"reference":"Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"reference":"Soding J, Biegert A, Lupas AN. 2005. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Research 33: W244-W248.
","pubmedId":"","doi":"10.1093/nar/gki408"},{"reference":"Zorawik M, Jacobs-Sera D, Freise AC, SEA-PHAGES, Reddi K. 2024. Isolation of Bacteriophages on Actinobacteria Hosts. Methods in Molecular Biology,Phage Engineering and Analysis : 273-298.
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