Mycobacteriophage Lilbit was isolated using Mycobacterium smegmatis mc²155. It has a genome consisting of 65,106 base pairs with 110 putative genes and GC content of 63.4%. Based on gene content similarity, it is assigned to actinobacteriophage cluster S. Functions were predicted for 40 genes. There were no tRNAs identified in the genome.
","acknowledgements":"We would like to acknowledge support from the HHMI Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program and the Pittsburgh Bacteriophage Institute. We acknowledge Shalee Page, Vic Sivanathan, Debbie Jacobs-Sera, Graham Hatfull, and Daniel A. Russell for their support which included genome sequencing and assembly, genome QC, and paper reviews. We also acknowledge Kate Aberger who discovered Lilbit when she was a student at Southern Connecticut State University.
","authors":[{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["writing_originalDraft","writing_reviewEditing","investigation"],"email":"ellie.clavette@ctstate.edu","firstName":"Ellie ","lastName":"Clavette","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern /Torrington High School"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"andrea.chuva@ctstate.edu","firstName":" Andrea ","lastName":"Chuva","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"nicholas.gruener@ctstate.edu","firstName":"Nicholas ","lastName":"Gruener","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"juniper.zinck@ctstate.edu","firstName":"Juniper","lastName":"Zinck","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["dataCuration","fundingAcquisition","project","resources","supervision","writing_reviewEditing"],"email":"sharon.gusky@ctstate.edu","firstName":"Sharon","lastName":"Gusky","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":null,"dataTable":null,"extendedData":[],"funding":"This material is based upon work supported by the National Science Foundation under Grants Number 1801062 and Number 2129896. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
","image":{"url":"https://portal.micropublication.org/uploads/603dd6ac56fcdc88861148cee884d156.jpg"},"imageCaption":"Lilbit produces plaques ~ 1 mm in diameter (A) and is composed of a capsid 69.81 +/- 3.13 nm in width with a tail 283.58 +/- 7.02 nm in length (B).
","imageTitle":"Plaques and Virion Morphology for Lilbit
","methods":"","reagents":"","patternDescription":"Bacteriophages are increasingly being developed for their potential use as therapeutic agents. In the case of mycobacteriophages, they have been used to treat extreme cases of infection in patients with cystic fibrosis, where Mycobacterium abscessus infection can be detrimental to the lungs, as well as in cases of infections by Mycobacterium avium and Mycobacterium chelonae. (Nick et al. 2022, Dedrick et al. 2023). Here we report on the isolation and characterization of a novel mycobacteriophage, Lilbit.
Lilbit was isolated from a sample of composting plant material collected in New Haven, Connecticut (GPS coordinates: 41.335200, -72.941760). The sample was resuspended in 7H9 liquid medium and inoculated with M. smegmatis mc2155. After incubation with shaking at 42˚C for several days, the culture was filtered and the filtrate plated in top agar with M. smegmatis, resulting in plaques of phage Lilbit, which were purified through multiple rounds of plating until consistent plaque morphology was obtained. Negative stain transmission electron microscopy revealed Lilbit to possess siphovirus morphology characterized by a non-contractile and flexible tail.
Lilbit DNA was extracted using the Promega Wizard DNA kit and sequenced by the Pittsburgh Bacteriophage Institute using an Illumina MiSeq (v3 reagents), with libraries prepped using the NEB Ultra II FS kit. This produced 195,422 single-end 150 base raw reads that were assembled using Newbler version 2.9 into a 65,106 base-pair genome with a shot-gun coverage of 419 (ref). The assembly and genome termini was checked and Consed V29, revealing an 11bp 3’ single-strand overhang of 5’-GCGCGCAGCGC at the termini (ref) Lilbit was assigned to cluster S based on have a gene content similarity of at least 35% to the phages already assigned to cluster S. (Pope et al., 2017).
The sequenced genome was auto-annotated using the Phage Evidence Collection And Annotation Network (PECAAN) v 20221109 (Rinehart et al., 2015) using Glimmer (Delcher et al., 2007) and GeneMark (Lukashin and Borodovsky 1998), with start sites manually refined with Phamerator using Actino draft database v578 (Cresawn etal., 2011) and Starterator (Pacey 2016). BLAST, using the Actinobacteriophage and NCBI non-redundant database (ref) and HHPRED, using the PDB_mmCIF70, Pfam- v.36, NCBI Conserved Domains databases (Söding 2005), and NCBIs Conserved Domains databases (Geer etal., 2015), were used to predict the gene functions. DeepTMHMM (Chaturvedi 2011) was used to determine if any putative genes coded for transmembrane proteins. Aragorn v1.2.38 (Laslett and Canback 2004.) and t-RNA scan SE v2.0 (Chan et al., 2021) were used to check for the presence of t-RNAs. Default settings were used for all software.
Lilbit was found to encode a total of 110 putative genes, 40 for which putative functions could be assigned. There were 96 genes transcribed in the one direction, and 14 transcribed in the other. No tRNAs were found. A programmed translational frameshift was identified for the genes which are predicted to code for tail assembly chaperones. These occur just before a 5,550 bp long gene predicted to encode the tape measure protein. Mycobacteriophage Lilbit is predicted to be lytic based on the absence of identifiable integrase or immunity repressor functions, consistent with other cluster S phages.
Nucleotide sequence accession numbers
Mycobacteriophage Lilbit is available at GenBank with Accession No. PV876982 and Sequence Read Archive (SRA) No. SRX29714289.
","references":[{"reference":"Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402.
","pubmedId":"","doi":""},{"reference":"Chan PP, Lin BY, Mak AJ, Lowe TM. 2021. tRNAscan-SE 2.0: improved detection and functional classification of transfer RNA genes. Nucleic Acids Res 49:9077–9096
","pubmedId":"","doi":""},{"reference":"Chaturvedi N, Shanker S, Singh VK, Sinha D, Pandey PN. 2011.“Hidden markov model for the prediction of transmembrane proteins using MATLAB”, Bioinformation, 2011;7(8):418-21. doi: 10.6026/97320630007418.8
","pubmedId":"","doi":""},{"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 12:395.
","pubmedId":"","doi":""},{"reference":"Dedrick RM, Guerrero-Bustamante CA, Garlena RA, Russell DA, Ford K, Harris K, Gilmour KC, Soothill J, Jacobs-Sera D, Schooley RT, Hatfull GF, Spencer H. 2019. Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus. Nat Med. doi: 10.1038/s41591-019-0437-z. Epub 2019 May 8. PMID: 31068712; PMCID: PMC6557439.
","pubmedId":"","doi":""},{"reference":"Dedrick, R. M., Smith, B. E., Cristinziano, M., Freeman, K. G., Jacobs-Sera, D., Belessis, Y., Whitney Brown, A., Cohen, K. A., Davidson, R. M., van Duin, D., Gainey, A., Garcia, C. B., Robert George, C. R., Haidar, G., Ip, W., Iredell, J., Khatami, A., Little, J. S., Malmivaara, K., & McMullan, B. J. (2022). Phage Therapy of Mycobacterium Infections: Compassionate-use of Phages in Twenty Patients with Drug-Resistant Mycobacterial Disease. Clinical Infectious Diseases, 76(1). https://doi.org/10.1093/cid/ciac453
","pubmedId":"","doi":""},{"reference":"Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23:673–679.
","pubmedId":"","doi":""},{"reference":"Geer RC, He J, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, and Bryant SH, 2015. “CDD: NCBI’s conserved domain database”. Nucleic Acids Res 43:D222–D226. https://doi.org/10.1093/nar/gku1221.
","pubmedId":"","doi":""},{"reference":"International Committee on Taxonomy of Viruses. 2009 Siphoviridae. ICTV. https://ictv.global/report_9th/dsDNA/Siphoviridae
","pubmedId":"","doi":""},{"reference":"Laslett D, Canback B. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res 32:11–16.
","pubmedId":"","doi":""},{"reference":"Lukashin AV, and Borodovsky M. 1998. “GeneMark.hmm: new solutions for gene finding”, Nucleic Acids Res 26:1107–1115, https://doi.org/10.1093/ nar/26.4.1107.
","pubmedId":"","doi":""},{"reference":"Nick, J. A., Dedrick, R. M., Gray, A. L., Vladar, E. K., Smith, B. E., Freeman, K. G., Malcolm, K. C., Epperson, L. E., Hasan, N. A., Hendrix, J., Callahan, K., Walton, K., Vestal, B., Wheeler, E., Rysavy, N. M., Poch, K., Caceres, S., Lovell, V. K., Hisert, K. B., & de Moura, V. C. (2022). Host and pathogen response to bacteriophage engineered against Mycobacterium abscessus lung infection. Cell, 185(11), 1860-1874.e12. https://doi.org/10.1016/j.cell.2022.04.024
","pubmedId":"","doi":""},{"reference":"Pacey M. 2016. Starterator guide, University of Pittsburgh, [Online], Available: https://seaphages.org/media/docs/Starterator_Guide_2016.pdf
","pubmedId":"","doi":""},{"reference":"Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Warner MH, Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES), Hatfull GF. 2017. Bacteriophages of Gordonia spp. display a spectrum of diversity and genetic relationships. mBio 8:e01069-17.
","pubmedId":"","doi":""},{"reference":"Rinehart C A, Gaffney BL, Smith JR, and Wood J D.2015, PECAAN: Phage Evidence Collection and Annotation Network. Western Kentucky University Bioinformatics and Information Science Center, [Online], Available: https://discover.kbrinsgd.org/login
","pubmedId":"","doi":""},{"reference":"Söding J, Biegert A, and Lupas AN.2005. “The HHpred interactive server for protein homology detection and structure prediction”, Nucleic Acids Res 33: W244–W248. https://doi.org/10.1093/nar/gki408.
","pubmedId":"","doi":""}],"title":"Genome Sequence of Mycobacteriophage Lilbit
","reviews":[{"reviewer":{"displayName":"Sarah Ball"},"openAcknowledgement":true,"status":{"submitted":true}},{"reviewer":{"displayName":"Kristen Butela"},"openAcknowledgement":true,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"a58f17e8-f864-480d-9a0a-3e2317e234b4","decision":"revise","abstract":"Mycobacteriophage Lilbit was isolated using Mycobacterium smegmatis mc²155. It has a genome consisting of 65,106 base pairs with 110 putative genes and GC content of 63.4%. Based on gene content similarity, it is assigned to actinobacteriophage cluster S. Functions were predicted for 40 genes. There were no tRNAs identified in the genome.
","acknowledgements":"We would like to acknowledge support from the HHMI Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program and the Pittsburgh Bacteriophage Institute. We acknowledge Shallee Page, Vic Sivanathan, Debbie Jacobs-Sera, Graham Hatfull, and Daniel A. Russell for their support which included genome sequencing and assembly, genome QC, and paper reviews. We also acknowledge Kate Aberger who discovered Lilbit when she was a student at Southern Connecticut State University.
","authors":[{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["writing_originalDraft","writing_reviewEditing","investigation"],"email":"ellie.clavette@ctstate.edu","firstName":"Ellie ","lastName":"Clavette","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern /Torrington High School"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"andrea.chuva@ctstate.edu","firstName":" Andrea ","lastName":"Chuva","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"nicholas.gruener@ctstate.edu","firstName":"Nicholas ","lastName":"Gruener","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"juniper.zinck@ctstate.edu","firstName":"Juniper","lastName":"Zinck","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["dataCuration","fundingAcquisition","project","resources","supervision","writing_reviewEditing"],"email":"sharon.gusky@ctstate.edu","firstName":"Sharon","lastName":"Gusky","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This material is based upon work supported by the National Science Foundation under Grants Number 1801062 and Number 2129896. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
","image":{"url":"https://portal.micropublication.org/uploads/603dd6ac56fcdc88861148cee884d156.jpg"},"imageCaption":"Lilbit produces plaques ~ 1 mm in diameter (A) and is composed of a capsid 69.81 +/- 3.13 nm in width with a tail 283.58 +/- 7.02 nm in length (B):note the dark specs are artifacts.
","imageTitle":"Plaques and Virion Morphology for Lilbit
","methods":"","reagents":"","patternDescription":"Bacteriophages are viruses that can infect and kill bacterial cells and have potential use as therapeutic agents. They have been used to treat an antibiotic resistant infection caused by Mycobacterium abscessus (1). Here we report on the Mycobacterium Phage Lilbit which was discovered by Kate Aberger while she was a student at Southern Connecticut State University in New Haven, CT. This phage was isolated from a soil sample taken from a very moist compost pile using an enriched isolation method. It was named Lilbit because it creates tiny plaques with a diameter of 1mm. It is a cluster S phage which infects hosts in the Mycobacterium genus of bacteria. Mycobacterium Phage Lilbit has a sophovirus morphology which are characterized as having thin, flexible tails that are built with six units and with the head and tails forming separately (2).
The phage’s genome was sequencing by the Pittsburgh Bacteriophage Institute using Illumina MiSeq sequencer. This produced single end reads with an average read length of 150 base-pairs and a shot gun coverage of 419. The genome length was determined to be 65,106 bases. A FASTA file was created and uploaded into the PhagesDB database (3).
The FASTA file was retrieved from PhagesDB and uploaded into The Phage Evidence Collection And Annotation Network (PECAAN) version 20221109 (4) for auto-annotation. The start and stops for each gene were determined using Glimmer Start(5), GeneMark(6), Phamerator(7), and Starterator (8). Coding capacity was determined using GeneMark (6). All of this information as well as the final score, z-score, gaps and overlaps of genes, and coding capacity were carefully considered when refining the annotation and finding the best start and stop of each gene. NCBI BLAST (9), HHPred (10), and NCBIs (11) conserved domain databases were used to determine the gene functions.
DeepTMHMM (12) was used to determine if any genes with unknown functions coded for transmembrane proteins. Aragorn v1.2.38 (13) and tRNA scan SE v2.0 (14) were used to check for the presence of tRNAs.
The bacteriophage Lilbit was found to have a total of 110 genes consisting of 40 genes coding for known proteins and 70 genes with unidentified functions. There were 96 rightward running genes and 14 leftward running genes. The GC content was 63.4%. No tRNAs were found.
The genome has an 11bp 3’sticky overhang of that contains a sequence of GCGCGCAGCGC, this is a characteristic shared by phages in the S cluster (3). Phages in Cluster S are lytic and this was confirmed in Lilbit by the presence of genes coding for Lysin A, Lysin B, and Holin. A programmed frameshift was identified between genes #45 and #46 which code for tail assembly chaperones. These occur just before Gene #47 which is 5550 bp long and codes for the tape measure protein.
Nucleotide sequence accession numbers
Mycobacteriophage Lilbit is available at GenBank with Accession No. PV876982 and Sequence Read Archive (SRA) No. SRX29714289.
","references":[{"reference":"Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402.
","pubmedId":"","doi":""},{"reference":"Chan PP, Lin BY, Mak AJ, Lowe TM. 2021. tRNAscan-SE 2.0: improved detection and functional classification of transfer RNA genes. Nucleic Acids Res 49:9077–9096
","pubmedId":"","doi":""},{"reference":"Chaturvedi N, Shanker S, Singh VK, Sinha D, Pandey PN. 2011.“Hidden markov model for the prediction of transmembrane proteins using MATLAB”, Bioinformation, 2011;7(8):418-21. doi: 10.6026/97320630007418.8
","pubmedId":"","doi":""},{"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 12:395.
","pubmedId":"","doi":""},{"reference":"Dedrick RM, Guerrero-Bustamante CA, Garlena RA, Russell DA, Ford K, Harris K, Gilmour KC, Soothill J, Jacobs-Sera D, Schooley RT, Hatfull GF, Spencer H. 2019. Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus. Nat Med. doi: 10.1038/s41591-019-0437-z. Epub 2019 May 8. PMID: 31068712; PMCID: PMC6557439.
","pubmedId":"","doi":""},{"reference":"Dedrick, R. M., Smith, B. E., Cristinziano, M., Freeman, K. G., Jacobs-Sera, D., Belessis, Y., Whitney Brown, A., Cohen, K. A., Davidson, R. M., van Duin, D., Gainey, A., Garcia, C. B., Robert George, C. R., Haidar, G., Ip, W., Iredell, J., Khatami, A., Little, J. S., Malmivaara, K., & McMullan, B. J. (2022). Phage Therapy of Mycobacterium Infections: Compassionate-use of Phages in Twenty Patients with Drug-Resistant Mycobacterial Disease. Clinical Infectious Diseases, 76(1). https://doi.org/10.1093/cid/ciac453
","pubmedId":"","doi":""},{"reference":"Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23:673–679.
","pubmedId":"","doi":""},{"reference":"Geer RC, He J, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, and Bryant SH, 2015. “CDD: NCBI’s conserved domain database”. Nucleic Acids Res 43:D222–D226. https://doi.org/10.1093/nar/gku1221.
","pubmedId":"","doi":""},{"reference":"International Committee on Taxonomy of Viruses. 2009 Siphoviridae. ICTV. https://ictv.global/report_9th/dsDNA/Siphoviridae
","pubmedId":"","doi":""},{"reference":"Laslett D, Canback B. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res 32:11–16.
","pubmedId":"","doi":""},{"reference":"Lukashin AV, and Borodovsky M. 1998. “GeneMark.hmm: new solutions for gene finding”, Nucleic Acids Res 26:1107–1115, https://doi.org/10.1093/ nar/26.4.1107.
","pubmedId":"","doi":""},{"reference":"Nick, J. A., Dedrick, R. M., Gray, A. L., Vladar, E. K., Smith, B. E., Freeman, K. G., Malcolm, K. C., Epperson, L. E., Hasan, N. A., Hendrix, J., Callahan, K., Walton, K., Vestal, B., Wheeler, E., Rysavy, N. M., Poch, K., Caceres, S., Lovell, V. K., Hisert, K. B., & de Moura, V. C. (2022). Host and pathogen response to bacteriophage engineered against Mycobacterium abscessus lung infection. Cell, 185(11), 1860-1874.e12. https://doi.org/10.1016/j.cell.2022.04.024
","pubmedId":"","doi":""},{"reference":"Pacey M. 2016. Starterator guide, University of Pittsburgh, [Online], Available: https://seaphages.org/media/docs/Starterator_Guide_2016.pdf
","pubmedId":"","doi":""},{"reference":"Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Warner MH, Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES), Hatfull GF. 2017. Bacteriophages of Gordonia spp. display a spectrum of diversity and genetic relationships. mBio 8:e01069-17.
","pubmedId":"","doi":""},{"reference":"Rinehart C A, Gaffney BL, Smith JR, and Wood J D.2015, PECAAN: Phage Evidence Collection and Annotation Network. Western Kentucky University Bioinformatics and Information Science Center, [Online], Available: https://discover.kbrinsgd.org/login
","pubmedId":"","doi":""},{"reference":"Söding J, Biegert A, and Lupas AN.2005. “The HHpred interactive server for protein homology detection and structure prediction”, Nucleic Acids Res 33: W244–W248. https://doi.org/10.1093/nar/gki408.
","pubmedId":"","doi":""}],"title":"Genome Sequence of Mycobacteriophage Lilbit
","reviews":[{"reviewer":{"displayName":"Kristen Butela"},"openAcknowledgement":true,"status":{"submitted":true}},{"reviewer":{"displayName":"Sarah Ball"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"739fcf7c-873a-4ce2-826e-6b3ab4a4b8ed","decision":"revise","abstract":"Mycobacteriophage Lilbit was isolated using Mycobacterium smegmatis mc²155. It has a genome consisting of 65,106 base pairs with 110 putative genes and GC content of 63.4%. Based on gene content similarity, it is assigned to actinobacteriophage cluster S. Functions were predicted for 40 genes. There were no tRNAs identified in the genome.
","acknowledgements":"We would like to acknowledge support from the HHMI Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program and the Pittsburgh Bacteriophage Institute. We acknowledge Shallee Page, Vic Sivanathan, Debbie Jacobs-Sera, Graham Hatfull, and Daniel A. Russell for their support which included genome sequencing and assembly, genome QC, and paper reviews. We also acknowledge Kate Aberger who discovered Lilbit when she was a student at Southern Connecticut State University.
","authors":[{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["writing_originalDraft","writing_reviewEditing","investigation"],"email":"ellie.clavette@ctstate.edu","firstName":"Ellie ","lastName":"Clavette","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern /Torrington High School"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"andrea.chuva@ctstate.edu","firstName":" Andrea ","lastName":"Chuva","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"nicholas.gruener@ctstate.edu","firstName":"Nicholas ","lastName":"Gruener","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"juniper.zinck@ctstate.edu","firstName":"Juniper","lastName":"Zinck","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["dataCuration","fundingAcquisition","project","resources","supervision","writing_reviewEditing"],"email":"sharon.gusky@ctstate.edu","firstName":"Sharon","lastName":"Gusky","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":" 0009-0006-8460-6190"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This material is based upon work supported by the National Science Foundation under Grants Number 1801062 and Number 2129896. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
","image":{"url":"https://portal.micropublication.org/uploads/603dd6ac56fcdc88861148cee884d156.jpg"},"imageCaption":"Lilbit produces plaques with halos that are ~ 1 mm in diameter (A) and is composed of a capsid 69.81 +/- 3.13 nm in width with a tail 283.58 +/- 7.02 nm in length (B):note the dark specs are artifacts.
","imageTitle":"Plaques and Virion Morphology for Lilbit
","methods":"","reagents":"","patternDescription":"Bacteriophages are increasingly being studied for their potential use as therapeutic agents. Mycobacteriophages have been used to treat antibiotic-resistant infections in patients with cystic fibrosis, where Mycobacterium abscessus infection can be detrimental to the lungs, as well as in cases of infections by Mycobacterium avium and Mycobacterium chelonae. (Nick et al. 2022, Dedrick et al. 2023). Here we report on the isolation and characterization of a novel mycobacteriophage, Lilbit.
Lilbit was isolated from a sample of composting plant material collected in New Haven, Connecticut (GPS coordinates: 41.335200, -72.941760). The sample was resuspended in 7H9 liquid medium and inoculated with M. smegmatis mc2155. After incubation with shaking at 42˚C for several days, the culture was filtered and the filtrate plated in top agar with M. smegmatis, resulting in plaques of phage Lilbit, which were purified through multiple rounds of plating until consistent tiny plaques measuring 1mm in size were obtained. Negative stain transmission electron microscopy using uranyl acetate stain revealed Lilbit to possess siphovirus morphology characterized by a non-contractile and flexible tail.
Lilbit's DNA was extracted using the Promega Wizard DNA kit and sequenced by the Pittsburgh Bacteriophage Institute using an Illumina MiSeq (v3 reagents), with libraries prepped using the NEB Ultra II FS kit. This produced 195,422 single-end 150 base raw reads that were assembled using Newbler version 2.9 into a 65,106 base-pair genome with a shot-gun coverage of 419. The assembly and genome termini was checked and Consed V29, revealing an 11bp 3’ single-strand overhang of 5’-GCGCGCAGCGC at the termini. Lilbit was assigned to cluster S based on have a gene content similarity of at least 35% to the phages already assigned to cluster S. (Pope et al., 2017).
The sequenced genome was auto-annotated using the Phage Evidence Collection And Annotation Network (PECAAN) v20221109 (Rinehart et al., 2015) using Glimmer v3.02 (Delcher et al., 2007) and GeneMark v4.28 (Lukashin and Borodovsky 1998), with start sites manually refined with Phamerator v606 using Actino draft database v578 (Cresawn etal., 2011) and Starterator v1.2 (Pacey 2016). BLAST, using the Actinobacteriophage and NCBI non-redundant database v2.2.18 (Russell and Hatfull 2017) and HHPRED, using the PDB_mmCIF70, Pfam- v36, NCBI Conserved Domains databases (Söding 2005), and NCBIs Conserved Domains databases (Geer etal., 2015), were used to predict the gene functions. DeepTMHMM v1.0 (Chaturvedi 2011) was used to determine if any putative genes coded for transmembrane proteins. Aragorn v1.2.38 (Laslett and Canback 2004) and tRNA scan SE v2.0 (Chan et al., 2021) were used to check for the presence of tRNAs. Default settings were used for all software.
Lilbit was found to encode a total of 110 putative genes, 40 for which putative functions could be assigned. There were 96 genes transcribed in the one direction, and 14 transcribed in the other. No tRNAs were found. A programmed -1 translational frameshift was identified for the genes which are predicted to code for tail assembly chaperones. These occur just before a 5,550 bp long gene predicted to encode the tape measure protein. Mycobacteriophage Lilbit is predicted to be lytic based on the absence of identifiable integrase or immunity repressor functions, consistent with other cluster S phages.
Nucleotide sequence accession numbers
Mycobacteriophage Lilbit is available at GenBank with Accession No. PV876982 and Sequence Read Archive (SRA) No. SRX29714289.
","references":[{"reference":"Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402.
","pubmedId":"","doi":""},{"reference":"Chan PP, Lin BY, Mak AJ, Lowe TM. 2021. tRNAscan-SE 2.0: improved detection and functional classification of transfer RNA genes. Nucleic Acids Res 49:9077–9096
","pubmedId":"","doi":""},{"reference":"Chaturvedi N, Shanker S, Singh VK, Sinha D, Pandey PN. 2011.“Hidden markov model for the prediction of transmembrane proteins using MATLAB”, Bioinformation, 2011;7(8):418-21. doi: 10.6026/97320630007418.8
","pubmedId":"","doi":""},{"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 12:395.
","pubmedId":"","doi":""},{"reference":"Dedrick RM, Guerrero-Bustamante CA, Garlena RA, Russell DA, Ford K, Harris K, Gilmour KC, Soothill J, Jacobs-Sera D, Schooley RT, Hatfull GF, Spencer H. 2019. Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus. Nat Med. doi: 10.1038/s41591-019-0437-z. Epub 2019 May 8. PMID: 31068712; PMCID: PMC6557439.
","pubmedId":"","doi":""},{"reference":"Dedrick, R. M., Smith, B. E., Cristinziano, M., Freeman, K. G., Jacobs-Sera, D., Belessis, Y., Whitney Brown, A., Cohen, K. A., Davidson, R. M., van Duin, D., Gainey, A., Garcia, C. B., Robert George, C. R., Haidar, G., Ip, W., Iredell, J., Khatami, A., Little, J. S., Malmivaara, K., & McMullan, B. J. (2022). Phage Therapy of Mycobacterium Infections: Compassionate-use of Phages in Twenty Patients with Drug-Resistant Mycobacterial Disease. Clinical Infectious Diseases, 76(1). https://doi.org/10.1093/cid/ciac453
","pubmedId":"","doi":""},{"reference":"Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23:673–679.
","pubmedId":"","doi":""},{"reference":"Geer RC, He J, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, and Bryant SH, 2015. “CDD: NCBI’s conserved domain database”. Nucleic Acids Res 43:D222–D226. https://doi.org/10.1093/nar/gku1221.
","pubmedId":"","doi":""},{"reference":"International Committee on Taxonomy of Viruses. 2009 Siphoviridae. ICTV. https://ictv.global/report_9th/dsDNA/Siphoviridae
","pubmedId":"","doi":""},{"reference":"Laslett D, Canback B. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res 32:11–16.
","pubmedId":"","doi":""},{"reference":"Lukashin AV, and Borodovsky M. 1998. “GeneMark.hmm: new solutions for gene finding”, Nucleic Acids Res 26:1107–1115, https://doi.org/10.1093/ nar/26.4.1107.
","pubmedId":"","doi":""},{"reference":"Nick, J. A., Dedrick, R. M., Gray, A. L., Vladar, E. K., Smith, B. E., Freeman, K. G., Malcolm, K. C., Epperson, L. E., Hasan, N. A., Hendrix, J., Callahan, K., Walton, K., Vestal, B., Wheeler, E., Rysavy, N. M., Poch, K., Caceres, S., Lovell, V. K., Hisert, K. B., & de Moura, V. C. (2022). Host and pathogen response to bacteriophage engineered against Mycobacterium abscessus lung infection. Cell, 185(11), 1860-1874.e12. https://doi.org/10.1016/j.cell.2022.04.024
","pubmedId":"","doi":""},{"reference":"Pacey M. 2016. Starterator guide, University of Pittsburgh, [Online], Available: https://seaphages.org/media/docs/Starterator_Guide_2016.pdf
","pubmedId":"","doi":""},{"reference":"Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Warner MH, Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES), Hatfull GF. 2017. Bacteriophages of Gordonia spp. display a spectrum of diversity and genetic relationships. mBio 8:e01069-17.
","pubmedId":"","doi":""},{"reference":"Rinehart C A, Gaffney BL, Smith JR, and Wood J D.2015, PECAAN: Phage Evidence Collection and Annotation Network. Western Kentucky University Bioinformatics and Information Science Center, [Online], Available: https://discover.kbrinsgd.org/login
","pubmedId":"","doi":""},{"reference":"Russell, D A, Hatfull G F, 2017 “PhagesDB: the actinobacteriophage database”, Bioinformatics, Volume 33, Issue 5, March 2017, Pages784-786, https://doi.org/10.1093/bioinformatics/btw711
","pubmedId":"","doi":""},{"reference":"Söding J, Biegert A, and Lupas AN.2005. “The HHpred interactive server for protein homology detection and structure prediction”, Nucleic Acids Res 33: W244–W248. https://doi.org/10.1093/nar/gki408.
","pubmedId":"","doi":""}],"title":"Genome Sequence of Mycobacteriophage Lilbit
","reviews":[{"reviewer":{"displayName":"Sarah Ball"},"openAcknowledgement":false,"status":{"submitted":true}},{"reviewer":{"displayName":"Kristen Butela"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"0dcdb622-87e0-436d-a30e-7aa66def95d2","decision":"accept","abstract":"Mycobacteriophage Lilbit was isolated using Mycobacterium smegmatis mc²155. It has a genome consisting of 65,106 base pairs with 110 putative genes and GC content of 63.4%. Based on gene content similarity, it is assigned to actinobacteriophage cluster S. Functions were predicted for 40 genes. There were no tRNAs identified in the genome.
","acknowledgements":"We would like to acknowledge support from the HHMI Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program and the Pittsburgh Bacteriophage Institute. We acknowledge Shallee Page, Vic Sivanathan, Debbie Jacobs-Sera, Graham Hatfull, and Daniel A. Russell for their support which included genome sequencing and assembly, genome QC, and paper reviews. We also acknowledge Kate Aberger who discovered Lilbit when she was a student at Southern Connecticut State University.
","authors":[{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["writing_originalDraft","writing_reviewEditing","investigation"],"email":"ellie.clavette@ctstate.edu","firstName":"Ellie ","lastName":"Clavette","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern /Torrington High School"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"andrea.chuva@ctstate.edu","firstName":" Andrea ","lastName":"Chuva","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"nicholas.gruener@ctstate.edu","firstName":"Nicholas ","lastName":"Gruener","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"juniper.zinck@ctstate.edu","firstName":"Juniper","lastName":"Zinck","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["dataCuration","fundingAcquisition","project","resources","supervision","writing_reviewEditing"],"email":"sharon.gusky@ctstate.edu","firstName":"Sharon","lastName":"Gusky","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":" 0009-0006-8460-6190"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This material is based upon work supported by the National Science Foundation under Grants Number 1801062 and Number 2129896. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
","image":{"url":"https://portal.micropublication.org/uploads/603dd6ac56fcdc88861148cee884d156.jpg"},"imageCaption":"Lilbit produces plaques with halos that are ~ 1 mm in diameter (A) and is composed of a capsid 69.81 +/- 3.13 nm in width with a tail 283.58 +/- 7.02 nm in length (B):note the dark specs are artifacts.
","imageTitle":"Plaques and Virion Morphology for Lilbit
","methods":"","reagents":"","patternDescription":"Bacteriophages are increasingly being studied for their potential use as therapeutic agents. Mycobacteriophages have been used to treat antibiotic-resistant infections in patients with cystic fibrosis, where Mycobacterium abscessus infection can be detrimental to the lungs, as well as in cases of infections by Mycobacterium avium and Mycobacterium chelonae. (Nick et al. 2022, Dedrick et al. 2023). Here we report on the isolation and characterization of a novel mycobacteriophage, Lilbit, which was isolated using Mycobacterium smegmatis mc2155 as a host. There are a number of disease-causing organisms in the Mycobacterium genus including multi-drug resistant strains of M. tuberculosis and M. leprae which cause tuberculosis and leprosy, respectively. (Garg etal., 2017) Some bacteriophages isolated using Mycobacterium smegmatis will be able to infect other members of the genus. (Poxleitner 2018)
Lilbit was isolated from a sample of composting plant material collected in New Haven, Connecticut (GPS coordinates: 41.335200, -72.941760) using an enriched isolation method. The sample was resuspended in 7H9 liquid medium and inoculated with M. smegmatis mc2155. After incubation with shaking at 42˚C for several days, the culture was filtered and the filtrate plated in top agar with M. smegmatis, resulting in plaques of phage Lilbit, which were purified through multiple rounds of plating until consistent tiny plaques measuring 1mm in size were obtained. Negative stain transmission electron microscopy using uranyl acetate stain revealed Lilbit to possess siphovirus morphology characterized by a non-contractile and flexible tail.
Lilbit's DNA was extracted using the Promega Wizard DNA kit and sequenced by the Pittsburgh Bacteriophage Institute using an Illumina MiSeq (v3 reagents), with libraries prepped using the NEB Ultra II FS kit. This produced 195,422 single-end 150 base raw reads that were assembled using Newbler version 2.9 into a 65,106 base-pair genome with a shot-gun coverage of 419. The assembly and genome termini was checked using Consed V29 (Gordon and Green, 1988), revealing an 11bp 3’ single-strand overhang of 5’-GCGCGCAGCGC at the termini. Lilbit was assigned to cluster S based on having a gene content similarity of at least 35% to the phages already assigned to cluster S. (Pope et al., 2017). Like other cluster S phages Lilbit has putative HNH endonucleases, putative methyltransferase, and multiple putative glycosyltransferases. (Sevcik et al., 2023)
The sequenced genome was auto-annotated using the Phage Evidence Collection And Annotation Network (PECAAN) v20221109 (Rinehart et al., 2015) using Glimmer v3.02 (Delcher et al., 2007) and GeneMark v4.28 (Lukashin and Borodovsky 1998), with start sites manually refined with Phamerator v606 using Actino draft database v578 (Cresawn etal., 2011) and Starterator v1.2 (Pacey 2016). BLAST, using the Actinobacteriophage BLAST program (Russell and Hatfull 2017) and NCBI non-redundant database v2.2.18 (Altschul et al., 1997) and HHPRED, using the PDB_mmCIF70, Pfam- v36, NCBI Conserved Domains databases (Söding 2005), and NCBIs Conserved Domains databases (Geer et al., 2015), were used to predict the gene functions. DeepTMHMM v1.0 (Chaturvedi 2011) was used to determine if any putative genes coded for transmembrane proteins. Aragorn v1.2.38 (Laslett and Canback 2004) and tRNA scan SE v2.0 (Chan et al., 2021) were used to check for the presence of tRNAs. Default settings were used for all software.
Lilbit was found to encode a total of 110 putative genes, 40 for which putative functions could be assigned. There were 96 genes transcribed in the forward direction, and 14 transcribed in the reverse direction. No tRNAs were found. A programmed -1 translational frameshift was identified for the genes which are predicted to code for tail assembly chaperones. These occur just before a 5,550 bp long gene predicted to encode the tape measure protein. Mycobacteriophage Lilbit is predicted to be lytic based on the absence of identifiable integrase or immunity repressor functions, consistent with other cluster S phages.
Nucleotide sequence accession numbers
Mycobacteriophage Lilbit is available at GenBank with Accession No. PV876982 and Sequence Read Archive (SRA) No. SRX29714289.
","references":[{"reference":"Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402.
","pubmedId":"","doi":""},{"reference":"Chan PP, Lin BY, Mak AJ, Lowe TM. 2021. tRNAscan-SE 2.0: improved detection and functional classification of transfer RNA genes. Nucleic Acids Res 49:9077–9096
","pubmedId":"","doi":""},{"reference":"Chaturvedi N, Shanker S, Singh VK, Sinha D, Pandey PN. 2011.“Hidden markov model for the prediction of transmembrane proteins using MATLAB”, Bioinformation, 2011;7(8):418-21. doi: 10.6026/97320630007418.8
","pubmedId":"","doi":""},{"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 12:395.
","pubmedId":"","doi":""},{"reference":"Dedrick RM, Guerrero-Bustamante CA, Garlena RA, Russell DA, Ford K, Harris K, Gilmour KC, Soothill J, Jacobs-Sera D, Schooley RT, Hatfull GF, Spencer H. 2019. Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus. Nat Med. doi: 10.1038/s41591-019-0437-z. Epub 2019 May 8. PMID: 31068712; PMCID: PMC6557439.
","pubmedId":"","doi":""},{"reference":"Dedrick, R. M., Smith, B. E., Cristinziano, M., Freeman, K. G., Jacobs-Sera, D., Belessis, Y., Whitney Brown, A., Cohen, K. A., Davidson, R. M., van Duin, D., Gainey, A., Garcia, C. B., Robert George, C. R., Haidar, G., Ip, W., Iredell, J., Khatami, A., Little, J. S., Malmivaara, K., & McMullan, B. J. (2022). Phage Therapy of Mycobacterium Infections: Compassionate-use of Phages in Twenty Patients with Drug-Resistant Mycobacterial Disease. Clinical Infectious Diseases, 76(1). https://doi.org/10.1093/cid/ciac453
","pubmedId":"","doi":""},{"reference":"Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23:673–679.
","pubmedId":"","doi":""},{"reference":"Garg K, Gupta R, Bhalla M, Janmeja A. 2017. Multidrug-resistant tuberculosis and leprosy: An unsolved mystery. Lung India 34: 364.
","pubmedId":"","doi":"10.4103/lungindia.lungindia_451_15"},{"reference":"Geer RC, He J, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, and Bryant SH, 2015. “CDD: NCBI’s conserved domain database”. Nucleic Acids Res 43:D222–D226. https://doi.org/10.1093/nar/gku1221.
","pubmedId":"","doi":""},{"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":"International Committee on Taxonomy of Viruses. 2009 Siphoviridae. ICTV. https://ictv.global/report_9th/dsDNA/Siphoviridae
","pubmedId":"","doi":""},{"reference":"Laslett D, Canback B. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res 32:11–16.
","pubmedId":"","doi":""},{"reference":"Lukashin AV, and Borodovsky M. 1998. “GeneMark.hmm: new solutions for gene finding”, Nucleic Acids Res 26:1107–1115, https://doi.org/10.1093/ nar/26.4.1107.
","pubmedId":"","doi":""},{"reference":"Nick, J. A., Dedrick, R. M., Gray, A. L., Vladar, E. K., Smith, B. E., Freeman, K. G., Malcolm, K. C., Epperson, L. E., Hasan, N. A., Hendrix, J., Callahan, K., Walton, K., Vestal, B., Wheeler, E., Rysavy, N. M., Poch, K., Caceres, S., Lovell, V. K., Hisert, K. B., & de Moura, V. C. (2022). Host and pathogen response to bacteriophage engineered against Mycobacterium abscessus lung infection. Cell, 185(11), 1860-1874.e12. https://doi.org/10.1016/j.cell.2022.04.024
","pubmedId":"","doi":""},{"reference":"Pacey M. 2016. Starterator guide, University of Pittsburgh, [Online], Available: https://seaphages.org/media/docs/Starterator_Guide_2016.pdf
","pubmedId":"","doi":""},{"reference":"Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Warner MH, Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES), Hatfull GF. 2017. Bacteriophages of Gordonia spp. display a spectrum of diversity and genetic relationships. mBio 8:e01069-17.
","pubmedId":"","doi":""},{"reference":"Poxleitner M, Pope W, Jacobs-Sera D, Sivanathan V, Hatfull GF. 2018. HHMI SEA-HAGES phage discovery guide. Howard Hughes Medical Institute, Chevy Chase, MD. https://seaphagesphagediscoveryguide.helpdocsonline.com/home.
","pubmedId":"","doi":""},{"reference":"Rinehart C A, Gaffney BL, Smith JR, and Wood J D.2015, PECAAN: Phage Evidence Collection and Annotation Network. Western Kentucky University Bioinformatics and Information Science Center, [Online], Available: https://discover.kbrinsgd.org/login
","pubmedId":"","doi":""},{"reference":"Russell, D A, Hatfull G F, 2017 “PhagesDB: the actinobacteriophage database”, Bioinformatics, Volume 33, Issue 5, March 2017, Pages784-786, https://doi.org/10.1093/bioinformatics/btw711
","pubmedId":"","doi":""},{"reference":"Sevcik K, Preston P, Aulner M, Noordewier B, Tolsma SS. 2023. Complete Genome Sequences of Cluster S Mycobacteriophages Beelzebub, Raela, and RedRaider77. Microbiology Resource Announcements 12: 10.1128/mra.01173-22.
","pubmedId":"","doi":"10.1128/mra.01173-22"},{"reference":"Söding J, Biegert A, and Lupas AN.2005. “The HHpred interactive server for protein homology detection and structure prediction”, Nucleic Acids Res 33: W244–W248. https://doi.org/10.1093/nar/gki408.
","pubmedId":"","doi":""}],"title":"Genome Sequence of Mycobacteriophage Lilbit
","reviews":[],"curatorReviews":[]},{"id":"19c95b4d-5b77-43ea-bef5-57adfb696bea","decision":"revise","abstract":"Mycobacteriophage Lilbit was isolated using Mycobacterium smegmatis mc²155. It has a genome consisting of 65,106 base pairs with 110 putative genes and GC content of 63.4%. Based on gene content similarity, it is assigned to actinobacteriophage cluster S. Functions were predicted for 40 genes. There were no tRNAs identified in the genome.
","acknowledgements":"We would like to acknowledge support from the HHMI Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program and the Pittsburgh Bacteriophage Institute. We acknowledge Shallee Page, Vic Sivanathan, Debbie Jacobs-Sera, Graham Hatfull, and Daniel A. Russell for their support which included genome sequencing and assembly, genome QC, and paper reviews. We also acknowledge Kate Aberger who discovered Lilbit when she was a student at Southern Connecticut State University.
","authors":[{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["writing_originalDraft","writing_reviewEditing","investigation"],"email":"ellie.clavette@ctstate.edu","firstName":"Ellie ","lastName":"Clavette","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern /Torrington High School"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"andrea.chuva@ctstate.edu","firstName":" Andrea ","lastName":"Chuva","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"nicholas.gruener@ctstate.edu","firstName":"Nicholas ","lastName":"Gruener","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"juniper.zinck@ctstate.edu","firstName":"Juniper","lastName":"Zinck","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["dataCuration","fundingAcquisition","project","resources","supervision","writing_reviewEditing"],"email":"sharon.gusky@ctstate.edu","firstName":"Sharon","lastName":"Gusky","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":" 0009-0006-8460-6190"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This material is based upon work supported by the National Science Foundation under Grants Number 1801062 and Number 2129896. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
","image":{"url":"https://portal.micropublication.org/uploads/603dd6ac56fcdc88861148cee884d156.jpg"},"imageCaption":"Lilbit produces plaques with halos that are ~ 1 mm in diameter (A) and is composed of a capsid 69.81 +/- 3.13 nm in width with a tail 283.58 +/- 7.02 nm in length (B):note the dark specs are artifacts.
","imageTitle":"Plaques and Virion Morphology for Lilbit
","methods":"","reagents":"","patternDescription":"Bacteriophages are increasingly being studied for their potential use as therapeutic agents. Mycobacteriophages have been used to treat antibiotic-resistant infections in patients with cystic fibrosis, where Mycobacterium abscessus infection can be detrimental to the lungs, as well as in cases of infections by Mycobacterium avium and Mycobacterium chelonae. (Nick et al. 2022, Dedrick et al. 2023). Here we report on the isolation and characterization of a novel mycobacteriophage, Lilbit, which was isolated using Mycobacterium smegmatis mc2155 as a host. There are a number of disease-causing organisms in the Mycobacterium genus including multi-drug resistant strains of M. tuberculosis and M. leprae which cause tuberculosis and leprosy, respectively. (Garg etal., 2017) Some bacteriophages isolated using Mycobacterium smegmatis will be able to infect other members of the genus. (Poxleitner 2018)
Lilbit was isolated from a sample of composting plant material collected in New Haven, Connecticut (GPS coordinates: 41.335200, -72.941760) using an enriched isolation method. The sample was resuspended in 7H9 liquid medium and inoculated with M. smegmatis mc2155. After incubation with shaking at 42˚C for several days, the culture was filtered and the filtrate plated in top agar with M. smegmatis, resulting in plaques of phage Lilbit, which were purified through multiple rounds of plating until consistent tiny plaques measuring 1mm in size were obtained. Negative stain transmission electron microscopy using uranyl acetate stain revealed Lilbit to possess siphovirus morphology characterized by a non-contractile and flexible tail.
Lilbit's DNA was extracted using the Promega Wizard DNA kit and sequenced by the Pittsburgh Bacteriophage Institute using an Illumina MiSeq (v3 reagents), with libraries prepped using the NEB Ultra II FS kit. This produced 195,422 single-end 150 base raw reads that were assembled using Newbler version 2.9 into a 65,106 base-pair genome with a shot-gun coverage of 419. The assembly and genome termini was checked using Consed V29 (Gordon and Green, 1988), revealing an 11bp 3’ single-strand overhang of 5’-GCGCGCAGCGC at the termini. Lilbit was assigned to cluster S based on having a gene content similarity of at least 35% to the phages already assigned to cluster S. (Pope et al., 2017). Like other cluster S phages Lilbit has putative HNH endonucleases, putative methyltransferase, and multiple putative glycosyltransferases. (Sevcik et al., 2023)
The sequenced genome was auto-annotated using the Phage Evidence Collection And Annotation Network (PECAAN) v20221109 (Rinehart et al., 2015) using Glimmer v3.02 (Delcher et al., 2007) and GeneMark v4.28 (Lukashin and Borodovsky 1998), with start sites manually refined with Phamerator v606 using Actino draft database v578 (Cresawn etal., 2011) and Starterator v1.2 (Pacey 2016). BLAST, using the Actinobacteriophage BLAST program (Russell and Hatfull 2017) and NCBI non-redundant database v2.2.18 (Altschul et al., 1997) and HHPRED, using the PDB_mmCIF70, Pfam- v36, NCBI Conserved Domains databases (Söding 2005), and NCBIs Conserved Domains databases (Geer et al., 2015), were used to predict the gene functions. DeepTMHMM v1.0 (Chaturvedi 2011) was used to determine if any putative genes coded for transmembrane proteins. Aragorn v1.2.38 (Laslett and Canback 2004) and tRNA scan SE v2.0 (Chan et al., 2021) were used to check for the presence of tRNAs. Default settings were used for all software.
Lilbit was found to encode a total of 110 putative genes, 40 for which putative functions could be assigned. There were 96 genes transcribed in the forward direction, and 14 transcribed in the reverse direction. No tRNAs were found. A programmed -1 translational frameshift was identified for the genes which are predicted to code for tail assembly chaperones. These occur just before a 5,550 bp long gene predicted to encode the tape measure protein. Mycobacteriophage Lilbit is predicted to be lytic based on the absence of identifiable integrase or immunity repressor functions, consistent with other cluster S phages.
Nucleotide sequence accession numbers
Mycobacteriophage Lilbit is available at GenBank with Accession No. PV876982 and Sequence Read Archive (SRA) No. SRX29714289.
","references":[{"reference":"Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402.
","pubmedId":"","doi":""},{"reference":"Chan PP, Lin BY, Mak AJ, Lowe TM. 2021. tRNAscan-SE 2.0: improved detection and functional classification of transfer RNA genes. Nucleic Acids Res 49:9077–9096
","pubmedId":"","doi":""},{"reference":"Chaturvedi N, Shanker S, Singh VK, Sinha D, Pandey PN. 2011.“Hidden markov model for the prediction of transmembrane proteins using MATLAB”, Bioinformation, 2011;7(8):418-21. doi: 10.6026/97320630007418.8
","pubmedId":"","doi":""},{"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 12:395.
","pubmedId":"","doi":""},{"reference":"Dedrick RM, Guerrero-Bustamante CA, Garlena RA, Russell DA, Ford K, Harris K, Gilmour KC, Soothill J, Jacobs-Sera D, Schooley RT, Hatfull GF, Spencer H. 2019. Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus. Nat Med. doi: 10.1038/s41591-019-0437-z. Epub 2019 May 8. PMID: 31068712; PMCID: PMC6557439.
","pubmedId":"","doi":""},{"reference":"Dedrick, R. M., Smith, B. E., Cristinziano, M., Freeman, K. G., Jacobs-Sera, D., Belessis, Y., Whitney Brown, A., Cohen, K. A., Davidson, R. M., van Duin, D., Gainey, A., Garcia, C. B., Robert George, C. R., Haidar, G., Ip, W., Iredell, J., Khatami, A., Little, J. S., Malmivaara, K., & McMullan, B. J. (2022). Phage Therapy of Mycobacterium Infections: Compassionate-use of Phages in Twenty Patients with Drug-Resistant Mycobacterial Disease. Clinical Infectious Diseases, 76(1). https://doi.org/10.1093/cid/ciac453
","pubmedId":"","doi":""},{"reference":"Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23:673–679.
","pubmedId":"","doi":""},{"reference":"Garg K, Gupta R, Bhalla M, Janmeja A. 2017. Multidrug-resistant tuberculosis and leprosy: An unsolved mystery. Lung India 34: 364.
","pubmedId":"","doi":"10.4103/lungindia.lungindia_451_15"},{"reference":"Geer RC, He J, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, and Bryant SH, 2015. “CDD: NCBI’s conserved domain database”. Nucleic Acids Res 43:D222–D226. https://doi.org/10.1093/nar/gku1221.
","pubmedId":"","doi":""},{"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":"International Committee on Taxonomy of Viruses. 2009 Siphoviridae. ICTV. https://ictv.global/report_9th/dsDNA/Siphoviridae
","pubmedId":"","doi":""},{"reference":"Laslett D, Canback B. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res 32:11–16.
","pubmedId":"","doi":""},{"reference":"Lukashin AV, and Borodovsky M. 1998. “GeneMark.hmm: new solutions for gene finding”, Nucleic Acids Res 26:1107–1115, https://doi.org/10.1093/ nar/26.4.1107.
","pubmedId":"","doi":""},{"reference":"Nick, J. A., Dedrick, R. M., Gray, A. L., Vladar, E. K., Smith, B. E., Freeman, K. G., Malcolm, K. C., Epperson, L. E., Hasan, N. A., Hendrix, J., Callahan, K., Walton, K., Vestal, B., Wheeler, E., Rysavy, N. M., Poch, K., Caceres, S., Lovell, V. K., Hisert, K. B., & de Moura, V. C. (2022). Host and pathogen response to bacteriophage engineered against Mycobacterium abscessus lung infection. Cell, 185(11), 1860-1874.e12. https://doi.org/10.1016/j.cell.2022.04.024
","pubmedId":"","doi":""},{"reference":"Pacey M. 2016. Starterator guide, University of Pittsburgh, [Online], Available: https://seaphages.org/media/docs/Starterator_Guide_2016.pdf
","pubmedId":"","doi":""},{"reference":"Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Warner MH, Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES), Hatfull GF. 2017. Bacteriophages of Gordonia spp. display a spectrum of diversity and genetic relationships. mBio 8:e01069-17.
","pubmedId":"","doi":""},{"reference":"Poxleitner M, Pope W, Jacobs-Sera D, Sivanathan V, Hatfull GF. 2018. HHMI SEA-HAGES phage discovery guide. Howard Hughes Medical Institute, Chevy Chase, MD. https://seaphagesphagediscoveryguide.helpdocsonline.com/home.
","pubmedId":"","doi":""},{"reference":"Rinehart C A, Gaffney BL, Smith JR, and Wood J D.2015, PECAAN: Phage Evidence Collection and Annotation Network. Western Kentucky University Bioinformatics and Information Science Center, [Online], Available: https://discover.kbrinsgd.org/login
","pubmedId":"","doi":""},{"reference":"Russell, D A, Hatfull G F, 2017 “PhagesDB: the actinobacteriophage database”, Bioinformatics, Volume 33, Issue 5, March 2017, Pages784-786, https://doi.org/10.1093/bioinformatics/btw711
","pubmedId":"","doi":""},{"reference":"Sevcik K, Preston P, Aulner M, Noordewier B, Tolsma SS. 2023. Complete Genome Sequences of Cluster S Mycobacteriophages Beelzebub, Raela, and RedRaider77. Microbiology Resource Announcements 12: 10.1128/mra.01173-22.
","pubmedId":"","doi":"10.1128/mra.01173-22"},{"reference":"Söding J, Biegert A, and Lupas AN.2005. “The HHpred interactive server for protein homology detection and structure prediction”, Nucleic Acids Res 33: W244–W248. https://doi.org/10.1093/nar/gki408.
","pubmedId":"","doi":""}],"title":"Genome Sequence of Mycobacteriophage Lilbit
","reviews":[],"curatorReviews":[]},{"id":"41b0111e-5472-458c-b19c-cdee3de3d1d7","decision":"edit","abstract":"Mycobacteriophage Lilbit was isolated using Mycobacterium smegmatis mc²155. It has a genome consisting of 65,106 base pairs with 110 putative genes and GC content of 63.4%. Based on gene content similarity, it is assigned to actinobacteriophage cluster S. Functions were predicted for 40 genes. There were no tRNAs identified in the genome.
","acknowledgements":"We would like to acknowledge support from the HHMI Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program and the Pittsburgh Bacteriophage Institute. We acknowledge Shallee Page, Vic Sivanathan, Debbie Jacobs-Sera, Graham Hatfull, and Daniel A. Russell for their support which included genome sequencing and assembly, genome QC, and paper reviews. We also acknowledge Kate Aberger who discovered Lilbit when she was a student at Southern Connecticut State University.
","authors":[{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["writing_originalDraft","writing_reviewEditing","investigation"],"email":"ellie.clavette@ctstate.edu","firstName":"Ellie ","lastName":"Clavette","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern /Torrington High School"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"andrea.chuva@ctstate.edu","firstName":" Andrea ","lastName":"Chuva","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"nicholas.gruener@ctstate.edu","firstName":"Nicholas ","lastName":"Gruener","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"juniper.zinck@ctstate.edu","firstName":"Juniper","lastName":"Zinck","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["dataCuration","fundingAcquisition","project","resources","supervision","writing_reviewEditing"],"email":"sharon.gusky@ctstate.edu","firstName":"Sharon","lastName":"Gusky","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":" 0009-0006-8460-6190"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This material is based upon work supported by the National Science Foundation under Grants Number 1801062 and Number 2129896. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
","image":{"url":"https://portal.micropublication.org/uploads/603dd6ac56fcdc88861148cee884d156.jpg"},"imageCaption":"Lilbit produces plaques with halos that are ~ 1 mm in diameter (A) and is composed of a capsid 69.81 +/- 3.13 nm in width with a tail 283.58 +/- 7.02 nm in length (B):note the dark specs are artifacts.
","imageTitle":"Plaques and Virion Morphology for Lilbit
","methods":"","reagents":"","patternDescription":"Bacteriophages are increasingly being studied for their potential use as therapeutic agents. Mycobacteriophages have been used to treat antibiotic-resistant infections in patients with cystic fibrosis, where Mycobacterium abscessus infection can be detrimental to the lungs, as well as in cases of infections by Mycobacterium avium and Mycobacterium chelonae. (Nick et al. 2022, Dedrick et al. 2023). Here we report on the isolation and characterization of a novel mycobacteriophage, Lilbit, which was isolated using Mycobacterium smegmatis mc2155 as a host. There are a number of disease-causing organisms in the Mycobacterium genus including multi-drug resistant strains of M. tuberculosis and M. leprae which cause tuberculosis and leprosy, respectively. (Garg etal., 2017) Some bacteriophages isolated using Mycobacterium smegmatis will be able to infect other members of the genus. (Poxleitner 2018)
Lilbit was isolated from a sample of composting plant material collected in New Haven, Connecticut (GPS coordinates: 41.335200, -72.941760) using an enriched isolation method. The sample was resuspended in 7H9 liquid medium and inoculated with M. smegmatis mc2155. After incubation with shaking at 42˚C for several days, the culture was filtered and the filtrate plated in top agar with M. smegmatis, resulting in plaques of phage Lilbit, which were purified through multiple rounds of plating until consistent tiny plaques measuring 1mm in size were obtained. Negative stain transmission electron microscopy using uranyl acetate stain revealed Lilbit to possess siphovirus morphology characterized by a non-contractile and flexible tail.
Lilbit's DNA was extracted using the Promega Wizard DNA kit and sequenced by the Pittsburgh Bacteriophage Institute using an Illumina MiSeq (v3 reagents), with libraries prepped using the NEB Ultra II FS kit. This produced 195,422 single-end 150 base raw reads that were assembled using Newbler version 2.9 into a 65,106 base-pair genome with a shot-gun coverage of 419. The assembly and genome termini was checked using Consed V29 (Gordon and Green, 1988), revealing an 11bp 3’ single-strand overhang of 5’-GCGCGCAGCGC at the termini. Lilbit was assigned to cluster S based on having a gene content similarity of at least 35% to the phages already assigned to cluster S. (Pope et al., 2017). Like other cluster S phages Lilbit has putative HNH endonucleases, putative methyltransferase, and multiple putative glycosyltransferases. (Sevcik et al., 2023)
The sequenced genome was auto-annotated using the Phage Evidence Collection And Annotation Network (PECAAN) v20221109 (Rinehart et al., 2015) using Glimmer v3.02 (Delcher et al., 2007) and GeneMark v4.28 (Lukashin and Borodovsky 1998), with start sites manually refined with Phamerator v606 using Actino draft database v578 (Cresawn etal., 2011) and Starterator v1.2 (Pacey 2016). BLAST, using the Actinobacteriophage BLAST program (Russell and Hatfull 2017) and NCBI non-redundant database v2.2.18 (Altschul et al., 1997) and HHPRED, using the PDB_mmCIF70, Pfam- v36, NCBI Conserved Domains databases (Söding 2005), and NCBIs Conserved Domains databases (Geer et al., 2015), were used to predict the gene functions. DeepTMHMM v1.0 (Chaturvedi 2011) was used to determine if any putative genes coded for transmembrane proteins. Aragorn v1.2.38 (Laslett and Canback 2004) and tRNA scan SE v2.0 (Chan et al., 2021) were used to check for the presence of tRNAs. Default settings were used for all software.
Lilbit was found to encode a total of 110 putative genes, 40 for which putative functions could be assigned. There were 96 genes transcribed in the forward direction, and 14 transcribed in the reverse direction. No tRNAs were found. A programmed -1 translational frameshift was identified for the genes which are predicted to code for tail assembly chaperones. These occur just before a 5,550 bp long gene predicted to encode the tape measure protein. Mycobacteriophage Lilbit is predicted to be lytic based on the absence of identifiable integrase or immunity repressor functions, consistent with other cluster S phages.
Nucleotide sequence accession numbers
Mycobacteriophage Lilbit is available at GenBank with Accession No. PV876982 and Sequence Read Archive (SRA) No. SRX29714289.
","references":[{"reference":"Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402. doi: 10.1093/nar/25.17.3389. PMID: 9254694; PMCID: PMC146917.
","pubmedId":"","doi":""},{"reference":"Chan PP, Lin BY, Mak AJ, Lowe TM. 2021. tRNAscan-SE 2.0: improved detection and functional classification of transfer RNA genes. Nucleic Acids Res 49:9077–9096. doi: 10.1093/nar/gkab688. PMID: 34417604; PMCID: PMC8450103.
","pubmedId":"","doi":""},{"reference":"Chaturvedi N, Shanker S, Singh VK, Sinha D, Pandey PN. 2011.“Hidden markov model for the prediction of transmembrane proteins using MATLAB”, Bioinformation, 2011;7(8):418-21. doi: 10.6026/97320630007418.8
","pubmedId":"","doi":""},{"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 12:395. https://doi.org/10.1186/1471-2105-12-395
","pubmedId":"","doi":""},{"reference":"Dedrick RM, Guerrero-Bustamante CA, Garlena RA, Russell DA, Ford K, Harris K, Gilmour KC, Soothill J, Jacobs-Sera D, Schooley RT, Hatfull GF, Spencer H. 2019. Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus. Nat Med. doi: 10.1038/s41591-019-0437-z. Epub 2019 May 8. PMID: 31068712; PMCID: PMC6557439.
","pubmedId":"","doi":""},{"reference":"Dedrick, R. M., Smith, B. E., Cristinziano, M., Freeman, K. G., Jacobs-Sera, D., Belessis, Y., Whitney Brown, A., Cohen, K. A., Davidson, R. M., van Duin, D., Gainey, A., Garcia, C. B., Robert George, C. R., Haidar, G., Ip, W., Iredell, J., Khatami, A., Little, J. S., Malmivaara, K., & McMullan, B. J. (2022). Phage Therapy of Mycobacterium Infections: Compassionate-use of Phages in Twenty Patients with Drug-Resistant Mycobacterial Disease. Clinical Infectious Diseases, 76(1). https://doi.org/10.1093/cid/ciac453
","pubmedId":"","doi":""},{"reference":"Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23:673–679. doi: 10.1093/bioinformatics/btm009. PMID: 17237039; PMCID: PMC2387122.
","pubmedId":"","doi":""},{"reference":"Garg K, Gupta R, Bhalla M, Janmeja A. 2017. Multidrug-resistant tuberculosis and leprosy: An unsolved mystery. Lung India 34: 364.doi: 10.4103/lungindia.lungindia_451_15. PMID: 28671168; PMCID: PMC5504894.
","pubmedId":"","doi":"10.4103/lungindia.lungindia_451_15"},{"reference":"Geer RC, He J, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, and Bryant SH, 2015. “CDD: NCBI’s conserved domain database”. Nucleic Acids Res 43:D222–D226. https://doi.org/10.1093/nar/gku1221.
","pubmedId":"","doi":""},{"reference":"Gordon D, Abajian C, Green P. 1998. Consed: A Graphical Tool for Sequence Finishing. Genome Research 8: 195-202. https://doi.org/10.1101/gr.8.3.195 PMID: 9521923
","pubmedId":"","doi":"10.1101/gr.8.3.195"},{"reference":"International Committee on Taxonomy of Viruses. 2009 Siphoviridae. ICTV. https://ictv.global/report_9th/dsDNA/Siphoviridae
","pubmedId":"","doi":""},{"reference":"Laslett D, Canback B. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res 32:11–16. https://doi.org/10.1093/nar/gkh152
","pubmedId":"","doi":""},{"reference":"Lukashin AV, and Borodovsky M. 1998. “GeneMark.hmm: new solutions for gene finding”, Nucleic Acids Res 26:1107–1115, https://doi.org/10.1093/ nar/26.4.1107.
","pubmedId":"","doi":""},{"reference":"Nick, J. A., Dedrick, R. M., Gray, A. L., Vladar, E. K., Smith, B. E., Freeman, K. G., Malcolm, K. C., Epperson, L. E., Hasan, N. A., Hendrix, J., Callahan, K., Walton, K., Vestal, B., Wheeler, E., Rysavy, N. M., Poch, K., Caceres, S., Lovell, V. K., Hisert, K. B., & de Moura, V. C. (2022). Host and pathogen response to bacteriophage engineered against Mycobacterium abscessus lung infection. Cell, 185(11), 1860-1874.e12. https://doi.org/10.1016/j.cell.2022.04.024
","pubmedId":"","doi":""},{"reference":"Pacey M. 2016. Starterator guide, University of Pittsburgh, [Online], Available: https://seaphages.org/media/docs/Starterator_Guide_2016.pdf
","pubmedId":"","doi":""},{"reference":"Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Warner MH, Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES), Hatfull GF. 2017. Bacteriophages of Gordonia spp. display a spectrum of diversity and genetic relationships. mBio 8:e01069-17. doi: 10.1128/mBio.01069-17. PMID: 28811342; PMCID: PMC5559632.
","pubmedId":"","doi":""},{"reference":"Poxleitner M, Pope W, Jacobs-Sera D, Sivanathan V, Hatfull GF. 2018. HHMI SEA-HAGES phage discovery guide. Howard Hughes Medical Institute, Chevy Chase, MD. https://seaphagesphagediscoveryguide.helpdocsonline.com/home.
","pubmedId":"","doi":""},{"reference":"Rinehart C A, Gaffney BL, Smith JR, and Wood J D.2015, PECAAN: Phage Evidence Collection and Annotation Network. Western Kentucky University Bioinformatics and Information Science Center, [Online], Available: https://discover.kbrinsgd.org/login
","pubmedId":"","doi":""},{"reference":"Russell, D A, Hatfull G F, 2017 “PhagesDB: the actinobacteriophage database”, Bioinformatics, Volume 33, Issue 5, March 2017, Pages784-786, https://doi.org/10.1093/bioinformatics/btw711
","pubmedId":"","doi":""},{"reference":"Sevcik K, Preston P, Aulner M, Noordewier B, Tolsma SS. 2023. Complete Genome Sequences of Cluster S Mycobacteriophages Beelzebub, Raela, and RedRaider77. Microbiology Resource Announcements 12: 10.1128/mra.01173-22. doi: 10.1128/mra.01173-22. Epub 2022 Dec 12. PMID: 36507676; PMCID: PMC9872701.
","pubmedId":"","doi":"10.1128/mra.01173-22"},{"reference":"Söding J, Biegert A, and Lupas AN.2005. “The HHpred interactive server for protein homology detection and structure prediction”, Nucleic Acids Res 33: W244–W248. https://doi.org/10.1093/nar/gki408.
","pubmedId":"","doi":""}],"title":"Genome Sequence of Mycobacteriophage Lilbit
","reviews":[],"curatorReviews":[]},{"id":"1bbe5d8e-959d-404c-b66e-279fbf69a71b","decision":"publish","abstract":"Mycobacteriophage Lilbit was isolated using Mycobacterium smegmatis mc²155. It has a genome consisting of 65,106 base pairs with 110 putative genes and GC content of 63.4%. Based on gene content similarity, it is assigned to actinobacteriophage cluster S. Functions were predicted for 40 genes. There were no tRNAs identified in the genome.
","acknowledgements":"We would like to acknowledge support from the HHMI Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program and the Pittsburgh Bacteriophage Institute. We acknowledge Shallee Page, Vic Sivanathan, Debbie Jacobs-Sera, Graham Hatfull, and Daniel A. Russell for their support which included genome sequencing and assembly, genome QC, and paper reviews. We also acknowledge Kate Aberger who discovered Lilbit when she was a student at Southern Connecticut State University.
","authors":[{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["writing_originalDraft","writing_reviewEditing","investigation"],"email":"ellie.clavette@ctstate.edu","firstName":"Ellie ","lastName":"Clavette","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern /Torrington High School"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"andrea.chuva@ctstate.edu","firstName":" Andrea ","lastName":"Chuva","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"nicholas.gruener@ctstate.edu","firstName":"Nicholas ","lastName":"Gruener","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"juniper.zinck@ctstate.edu","firstName":"Juniper","lastName":"Zinck","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern"],"departments":["STEM"],"credit":["dataCuration","fundingAcquisition","project","resources","supervision","writing_reviewEditing"],"email":"sharon.gusky@ctstate.edu","firstName":"Sharon","lastName":"Gusky","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":" 0009-0006-8460-6190"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This material is based upon work supported by the National Science Foundation under Grants Number 1801062 and Number 2129896. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
","image":{"url":"https://portal.micropublication.org/uploads/603dd6ac56fcdc88861148cee884d156.jpg"},"imageCaption":"Lilbit produces plaques with halos that are ~ 1 mm in diameter (A) and is composed of a capsid 69.81 +/- 3.13 nm in width with a tail 283.58 +/- 7.02 nm in length (B):note the dark specs are artifacts.
","imageTitle":"Plaques and Virion Morphology for Lilbit
","methods":"","reagents":"","patternDescription":"Bacteriophages are increasingly being studied for their potential use as therapeutic agents. Mycobacteriophages have been used to treat antibiotic-resistant infections in patients with cystic fibrosis, where Mycobacterium abscessus infection can be detrimental to the lungs, as well as in cases of infections by Mycobacterium avium and Mycobacterium chelonae. (Nick et al. 2022, Dedrick et al. 2023). Here we report on the isolation and characterization of a novel mycobacteriophage, Lilbit, which was isolated using Mycobacterium smegmatis mc2155 as a host. There are a number of disease-causing organisms in the Mycobacterium genus including multi-drug resistant strains of M. tuberculosis and M. leprae which cause tuberculosis and leprosy, respectively. (Garg etal., 2017) Some bacteriophages isolated using Mycobacterium smegmatis will be able to infect other members of the genus. (Poxleitner 2018)
Lilbit was isolated from a sample of composting plant material collected in New Haven, Connecticut (GPS coordinates: 41.335200, -72.941760) using an enriched isolation method. The sample was resuspended in 7H9 liquid medium and inoculated with M. smegmatis mc2155. After incubation with shaking at 42˚C for several days, the culture was filtered and the filtrate plated in top agar with M. smegmatis, resulting in plaques of phage Lilbit, which were purified through multiple rounds of plating until consistent tiny plaques measuring 1mm in size were obtained. Negative stain transmission electron microscopy using uranyl acetate stain revealed Lilbit to possess siphovirus morphology characterized by a non-contractile and flexible tail.
Lilbit's DNA was extracted using the Promega Wizard DNA kit and sequenced by the Pittsburgh Bacteriophage Institute using an Illumina MiSeq (v3 reagents), with libraries prepped using the NEB Ultra II FS kit. This produced 195,422 single-end 150 base raw reads that were assembled using Newbler version 2.9 into a 65,106 base-pair genome with a shot-gun coverage of 419. The assembly and genome termini was checked using Consed V29 (Gordon and Green, 1988), revealing an 11bp 3’ single-strand overhang of 5’-GCGCGCAGCGC at the termini. Lilbit was assigned to cluster S based on having a gene content similarity of at least 35% to the phages already assigned to cluster S. (Pope et al., 2017). Like other cluster S phages Lilbit has putative HNH endonucleases, putative methyltransferase, and multiple putative glycosyltransferases. (Sevcik et al., 2023)
The sequenced genome was auto-annotated using the Phage Evidence Collection And Annotation Network (PECAAN) v20221109 (Rinehart et al., 2015) using Glimmer v3.02 (Delcher et al., 2007) and GeneMark v4.28 (Lukashin and Borodovsky 1998), with start sites manually refined with Phamerator v606 using Actino draft database v578 (Cresawn etal., 2011) and Starterator v1.2 (Pacey 2016). BLAST, using the Actinobacteriophage BLAST program (Russell and Hatfull 2017) and NCBI non-redundant database v2.2.18 (Altschul et al., 1997) and HHPRED, using the PDB_mmCIF70, Pfam- v36, NCBI Conserved Domains databases (Söding 2005), and NCBIs Conserved Domains databases (Geer et al., 2015), were used to predict the gene functions. DeepTMHMM v1.0 (Chaturvedi 2011) was used to determine if any putative genes coded for transmembrane proteins. Aragorn v1.2.38 (Laslett and Canback 2004) and tRNA scan SE v2.0 (Chan et al., 2021) were used to check for the presence of tRNAs. Default settings were used for all software.
Lilbit was found to encode a total of 110 putative genes, 40 for which putative functions could be assigned. There were 96 genes transcribed in the forward direction, and 14 transcribed in the reverse direction. No tRNAs were found. A programmed -1 translational frameshift was identified for the genes which are predicted to code for tail assembly chaperones. These occur just before a 5,550 bp long gene predicted to encode the tape measure protein. Mycobacteriophage Lilbit is predicted to be lytic based on the absence of identifiable integrase or immunity repressor functions, consistent with other cluster S phages.
Nucleotide sequence accession numbers
Mycobacteriophage Lilbit is available at GenBank with Accession No. PV876982 and Sequence Read Archive (SRA) No. SRX29714289.
","references":[{"reference":"Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17): 3389-402.
","pubmedId":"9254694","doi":""},{"reference":"Chan PP, Lin BY, Mak AJ, Lowe TM. 2021. tRNAscan-SE 2.0: improved detection and functional classification of transfer RNA genes. Nucleic Acids Res 49(16): 9077-9096.
","pubmedId":"34417604","doi":""},{"reference":"Chaturvedi N, Shanker S, Singh VK, Sinha D, Pandey PN. 2011.“Hidden markov model for the prediction of transmembrane proteins using MATLAB”, Bioinformation, 2011;7(8):418-21.
","pubmedId":"","doi":"10.6026/97320630007418.8"},{"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":"Dedrick RM, Guerrero-Bustamante CA, Garlena RA, Russell DA, Ford K, Harris K, et al., Spencer H. 2019. Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus. Nat Med 25(5): 730-733.
","pubmedId":"31068712","doi":""},{"reference":"Dedrick RM, Smith BE, Cristinziano M, Freeman KG, Jacobs-Sera D, Belessis Y, et al., Hatfull. 2022. Phage Therapy of Mycobacterium Infections: Compassionate Use of Phages in 20 Patients With Drug-Resistant Mycobacterial Disease. Clinical Infectious Diseases 76: 103-112.
","pubmedId":"","doi":"10.1093/cid/ciac453"},{"reference":"Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23(6): 673-9.
","pubmedId":"17237039","doi":""},{"reference":"Garg K, Gupta R, Bhalla M, Janmeja A. 2017. Multidrug-resistant tuberculosis and leprosy: An unsolved mystery. Lung India 34: 364.doi: 10.4103/lungindia.lungindia_451_15. PMID: 28671168; PMCID: PMC5504894.
","pubmedId":"","doi":"10.4103/lungindia.lungindia_451_15"},{"reference":"Geer RC, He J, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, and Bryant SH, 2015. “CDD: NCBI’s conserved domain database”. Nucleic Acids Res 43:D222–D226.
","pubmedId":"","doi":"10.1093/nar/gku1221"},{"reference":"Gordon D, Abajian C, Green P. 1998. Consed: A Graphical Tool for Sequence Finishing. Genome Research 8: 195-202. https://doi.org/10.1101/gr.8.3.195 PMID: 9521923
","pubmedId":"","doi":"10.1101/gr.8.3.195"},{"reference":"International Committee on Taxonomy of Viruses. 2009 Siphoviridae. ICTV. https://ictv.global/report_9th/dsDNA/Siphoviridae
","pubmedId":"","doi":""},{"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":"Lukashin A. 1998. GeneMark.hmm: new solutions for gene finding. Nucleic Acids Research 26: 1107-1115.
","pubmedId":"","doi":"10.1093/nar/26.4.1107"},{"reference":"Nick JA, Dedrick RM, Gray AL, Vladar EK, Smith BE, Freeman KG, et al., Davidson. 2022. Host and pathogen response to bacteriophage engineered against Mycobacterium abscessus lung infection. Cell 185: 1860-1874.e12.
","pubmedId":"","doi":"10.1016/j.cell.2022.04.024"},{"reference":"Pacey M. 2016. Starterator guide, University of Pittsburgh, [Online], Available: https://seaphages.org/media/docs/Starterator_Guide_2016.pdf
","pubmedId":"","doi":""},{"reference":"Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Hatfull GF. 2017. Bacteriophages of Gordonia spp. Display a Spectrum of Diversity and Genetic Relationships. mBio 8(4): 10.1128/mBio.01069-17.
","pubmedId":"28811342","doi":""},{"reference":"Poxleitner M, Pope W, Jacobs-Sera D, Sivanathan V, Hatfull GF. 2018. HHMI SEA-HAGES phage discovery guide. Howard Hughes Medical Institute, Chevy Chase, MD. https://seaphagesphagediscoveryguide.helpdocsonline.com/home.
","pubmedId":"","doi":""},{"reference":"Rinehart C A, Gaffney BL, Smith JR, and Wood J D.2015, PECAAN: Phage Evidence Collection and Annotation Network. Western Kentucky University Bioinformatics and Information Science Center, [Online], Available: https://discover.kbrinsgd.org/login
","pubmedId":"","doi":""},{"reference":"Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"reference":"Sevcik K, Preston P, Aulner M, Noordewier B, Tolsma SS. 2023. Complete Genome Sequences of Cluster S Mycobacteriophages Beelzebub, Raela, and RedRaider77. Microbiology Resource Announcements 12: 10.1128/mra.01173-22. doi: 10.1128/mra.01173-22. Epub 2022 Dec 12. PMID: 36507676; PMCID: PMC9872701.
","pubmedId":"","doi":"10.1128/mra.01173-22"},{"reference":"Söding J, Biegert A, and Lupas AN.2005. “The HHpred interactive server for protein homology detection and structure prediction”, Nucleic Acids Res 33: W244–W248.
","pubmedId":"","doi":"10.1093/nar/gki408"}],"title":"Genome Sequence of Mycobacteriophage Lilbit
","reviews":[],"curatorReviews":[]},{"id":"5fd49ecc-6ea1-4f70-af3f-7f559e1b0015","decision":"publish","abstract":"Mycobacteriophage Lilbit was isolated using Mycobacterium smegmatis mc²155. It has a genome consisting of 65,106 base pairs with 110 putative genes and GC content of 63.4%. Based on gene content similarity, it is assigned to actinobacteriophage cluster S. Functions were predicted for 40 genes. There were no tRNAs identified in the genome.
","acknowledgements":"We would like to acknowledge support from the HHMI Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program and the Pittsburgh Bacteriophage Institute. We acknowledge Shallee Page, Vic Sivanathan, Debbie Jacobs-Sera, Graham Hatfull, and Daniel A. Russell for their support which included genome sequencing and assembly, genome QC, and paper reviews. We also acknowledge Kate Aberger who discovered Lilbit when she was a student at Southern Connecticut State University.
","authors":[{"affiliations":["Connecticut State Community College Northwestern, Winsted, CT USA"],"departments":["STEM"],"credit":["writing_originalDraft","writing_reviewEditing","investigation"],"email":"ellie.clavette@ctstate.edu","firstName":"Ellie ","lastName":"Clavette","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern, Winsted, CT USA","Torrington High School, Torrington, CT USA"],"departments":["STEM",""],"credit":["investigation","writing_originalDraft"],"email":"andrea.chuva@ctstate.edu","firstName":" Andrea ","lastName":"Chuva","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern, Winsted, CT USA"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"nicholas.gruener@ctstate.edu","firstName":"Nicholas ","lastName":"Gruener","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern, Winsted, CT USA"],"departments":["STEM"],"credit":["investigation","writing_originalDraft"],"email":"juniper.zinck@ctstate.edu","firstName":"Juniper","lastName":"Zinck","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Connecticut State Community College Northwestern, Winsted, CT USA"],"departments":["STEM"],"credit":["dataCuration","fundingAcquisition","project","resources","supervision","writing_reviewEditing"],"email":"sharon.gusky@ctstate.edu","firstName":"Sharon","lastName":"Gusky","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":" 0009-0006-8460-6190"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This material is based upon work supported by the National Science Foundation under Grants Number 1801062 and Number 2129896. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
","image":{"url":"https://portal.micropublication.org/uploads/603dd6ac56fcdc88861148cee884d156.jpg"},"imageCaption":"Lilbit produces plaques with halos that are ~ 1 mm in diameter (A) and is composed of a capsid 69.81 +/- 3.13 nm in width with a tail 283.58 +/- 7.02 nm in length (B):note the dark specs are artifacts.
","imageTitle":"Plaques and Virion Morphology for Lilbit
","methods":"","reagents":"","patternDescription":"Bacteriophages are increasingly being studied for their potential use as therapeutic agents. Mycobacteriophages have been used to treat antibiotic-resistant infections in patients with cystic fibrosis, where Mycobacterium abscessus infection can be detrimental to the lungs, as well as in cases of infections by Mycobacterium avium and Mycobacterium chelonae. (Nick et al. 2022, Dedrick et al. 2023). Here we report on the isolation and characterization of a novel mycobacteriophage, Lilbit, which was isolated using Mycobacterium smegmatis mc2155 as a host. There are a number of disease-causing organisms in the Mycobacterium genus including multi-drug resistant strains of M. tuberculosis and M. leprae which cause tuberculosis and leprosy, respectively. (Garg etal., 2017) Some bacteriophages isolated using Mycobacterium smegmatis will be able to infect other members of the genus. (Poxleitner 2018)
Lilbit was isolated from a sample of composting plant material collected in New Haven, Connecticut (GPS coordinates: 41.335200, -72.941760) using an enriched isolation method. The sample was resuspended in 7H9 liquid medium and inoculated with M. smegmatis mc2155. After incubation with shaking at 42˚C for several days, the culture was filtered and the filtrate plated in top agar with M. smegmatis, resulting in plaques of phage Lilbit, which were purified through multiple rounds of plating until consistent tiny plaques measuring 1mm in size were obtained. Negative stain transmission electron microscopy using uranyl acetate stain revealed Lilbit to possess siphovirus morphology characterized by a non-contractile and flexible tail.
Lilbit's DNA was extracted using the Promega Wizard DNA kit and sequenced by the Pittsburgh Bacteriophage Institute using an Illumina MiSeq (v3 reagents), with libraries prepped using the NEB Ultra II FS kit. This produced 195,422 single-end 150 base raw reads that were assembled using Newbler version 2.9 into a 65,106 base-pair genome with a shot-gun coverage of 419. The assembly and genome termini was checked using Consed V29 (Gordon and Green, 1988), revealing an 11bp 3’ single-strand overhang of 5’-GCGCGCAGCGC at the termini. Lilbit was assigned to cluster S based on having a gene content similarity of at least 35% to the phages already assigned to cluster S. (Pope et al., 2017). Like other cluster S phages Lilbit has putative HNH endonucleases, putative methyltransferase, and multiple putative glycosyltransferases. (Sevcik et al., 2023)
The sequenced genome was auto-annotated using the Phage Evidence Collection And Annotation Network (PECAAN) v20221109 (Rinehart et al., 2015) using Glimmer v3.02 (Delcher et al., 2007) and GeneMark v4.28 (Lukashin and Borodovsky 1998), with start sites manually refined with Phamerator v606 using Actino draft database v578 (Cresawn etal., 2011) and Starterator v1.2 (Pacey 2016). BLAST, using the Actinobacteriophage BLAST program (Russell and Hatfull 2017) and NCBI non-redundant database v2.2.18 (Altschul et al., 1997) and HHPRED, using the PDB_mmCIF70, Pfam- v36, NCBI Conserved Domains databases (Söding 2005), and NCBIs Conserved Domains databases (Geer et al., 2015), were used to predict the gene functions. DeepTMHMM v1.0 (Chaturvedi 2011) was used to determine if any putative genes coded for transmembrane proteins. Aragorn v1.2.38 (Laslett and Canback 2004) and tRNA scan SE v2.0 (Chan et al., 2021) were used to check for the presence of tRNAs. Default settings were used for all software.
Lilbit was found to encode a total of 110 putative genes, 40 for which putative functions could be assigned. There were 96 genes transcribed in the forward direction, and 14 transcribed in the reverse direction. No tRNAs were found. A programmed -1 translational frameshift was identified for the genes which are predicted to code for tail assembly chaperones. These occur just before a 5,550 bp long gene predicted to encode the tape measure protein. Mycobacteriophage Lilbit is predicted to be lytic based on the absence of identifiable integrase or immunity repressor functions, consistent with other cluster S phages.
Nucleotide sequence accession numbers
Mycobacteriophage Lilbit is available at GenBank with Accession No. PV876982 and Sequence Read Archive (SRA) No. SRX29714289.
","references":[{"reference":"Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17): 3389-402.
","pubmedId":"9254694","doi":""},{"reference":"Chan PP, Lin BY, Mak AJ, Lowe TM. 2021. tRNAscan-SE 2.0: improved detection and functional classification of transfer RNA genes. Nucleic Acids Res 49(16): 9077-9096.
","pubmedId":"34417604","doi":""},{"reference":"Chaturvedi N, Shanker S, Singh VK, Sinha D, Pandey PN. 2011.“Hidden markov model for the prediction of transmembrane proteins using MATLAB”, Bioinformation, 2011;7(8):418-21.
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","pubmedId":"","doi":"10.1186/1471-2105-12-395"},{"reference":"Dedrick RM, Guerrero-Bustamante CA, Garlena RA, Russell DA, Ford K, Harris K, et al., Spencer H. 2019. Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus. Nat Med 25(5): 730-733.
","pubmedId":"31068712","doi":""},{"reference":"Dedrick RM, Smith BE, Cristinziano M, Freeman KG, Jacobs-Sera D, Belessis Y, et al., Hatfull. 2022. Phage Therapy of Mycobacterium Infections: Compassionate Use of Phages in 20 Patients With Drug-Resistant Mycobacterial Disease. Clinical Infectious Diseases 76: 103-112.
","pubmedId":"","doi":"10.1093/cid/ciac453"},{"reference":"Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23(6): 673-9.
","pubmedId":"17237039","doi":""},{"reference":"Garg K, Gupta R, Bhalla M, Janmeja A. 2017. Multidrug-resistant tuberculosis and leprosy: An unsolved mystery. Lung India 34: 364.doi: 10.4103/lungindia.lungindia_451_15. PMID: 28671168; PMCID: PMC5504894.
","pubmedId":"","doi":"10.4103/lungindia.lungindia_451_15"},{"reference":"Geer RC, He J, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, and Bryant SH, 2015. “CDD: NCBI’s conserved domain database”. Nucleic Acids Res 43:D222–D226.
","pubmedId":"","doi":"10.1093/nar/gku1221"},{"reference":"Gordon D, Abajian C, Green P. 1998. Consed: A Graphical Tool for Sequence Finishing. Genome Research 8: 195-202. https://doi.org/10.1101/gr.8.3.195 PMID: 9521923
","pubmedId":"","doi":"10.1101/gr.8.3.195"},{"reference":"International Committee on Taxonomy of Viruses. 2009 Siphoviridae. ICTV. https://ictv.global/report_9th/dsDNA/Siphoviridae
","pubmedId":"","doi":""},{"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":"Lukashin A. 1998. GeneMark.hmm: new solutions for gene finding. Nucleic Acids Research 26: 1107-1115.
","pubmedId":"","doi":"10.1093/nar/26.4.1107"},{"reference":"Nick JA, Dedrick RM, Gray AL, Vladar EK, Smith BE, Freeman KG, et al., Davidson. 2022. Host and pathogen response to bacteriophage engineered against Mycobacterium abscessus lung infection. Cell 185: 1860-1874.e12.
","pubmedId":"","doi":"10.1016/j.cell.2022.04.024"},{"reference":"Pacey M. 2016. Starterator guide, University of Pittsburgh, [Online], Available: https://seaphages.org/media/docs/Starterator_Guide_2016.pdf
","pubmedId":"","doi":""},{"reference":"Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Hatfull GF. 2017. Bacteriophages of Gordonia spp. Display a Spectrum of Diversity and Genetic Relationships. mBio 8(4): 10.1128/mBio.01069-17.
","pubmedId":"28811342","doi":""},{"reference":"Poxleitner M, Pope W, Jacobs-Sera D, Sivanathan V, Hatfull GF. 2018. HHMI SEA-HAGES phage discovery guide. Howard Hughes Medical Institute, Chevy Chase, MD. https://seaphagesphagediscoveryguide.helpdocsonline.com/home.
","pubmedId":"","doi":""},{"reference":"Rinehart C A, Gaffney BL, Smith JR, and Wood J D.2015, PECAAN: Phage Evidence Collection and Annotation Network. Western Kentucky University Bioinformatics and Information Science Center, [Online], Available: https://discover.kbrinsgd.org/login
","pubmedId":"","doi":""},{"reference":"Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
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","pubmedId":"","doi":"10.1128/mra.01173-22"},{"reference":"Söding J, Biegert A, and Lupas AN.2005. “The HHpred interactive server for protein homology detection and structure prediction”, Nucleic Acids Res 33: W244–W248.
","pubmedId":"","doi":"10.1093/nar/gki408"}],"title":"Genome Sequence of Mycobacteriophage Lilbit
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