Complete Genome Sequences of Cluster A Mycobacteriophages BobSwaget, Fred313, KADY, Lokk, MyraDee, Stagni, and StepMih

Butela, Kristen; Gurney, Susan M. R.; Hendrickson, Heather; LeBlanc‐Straceski, Janine M.; Zimmerman, Anastasia M.; Conant, Stephanie B.; Freed, Nikki E.; Silander, Olin K.; Thomson, Joshua J.; Berkes, Charlotte A.; Bertolez, Cristina; Davies, Courtney G.; Elinsky, Amber; Hanlon, Alison; Nersesyan, J.; Patel, Payal M.; Sherwood, John R.; Ngo, Tiffany Tieu; Wisniewski, Kathryn; Yacoo, Kathrine; Arendse, Paul M.; Bowlen, Nathan W.; Cunmulaj, Jasmina; Downs, Julie L.; Ferrenberg, Charlee A.; Gassman, Alexandra E.; Gilligan, Cody E. R.; Gorkiewicz, Emily; Harness, Christopher; Huffman, Anthony; Jones, Christina; Julien, Anna; Kupic, Alexis E.; Latu, S. F.; Manning, Thomas J.; Maxwell, Danielle N.; Meyer, C.; Reardon, Madeleine; Slaughter, Matthew J.; Swasey, Royce; Tennent, Rebecca; Torres, Victoria; Waller, Tamia; Worcester, Rachel M.; Yost, Brooke L.; Cresawn, Steven G.; Garlena, Rebecca A.; Jacobs-Sera, Deborah; Pope, Welkin H.; Russell, Daniel A.; Hatfull, Graham F.; Kagey, Jacob D.

doi:10.1128/genomea.01182-17

Cited by 4 publications

(4 citation statements)

References 22 publications

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“…Because both the integration cassettes of subcluster A2 phages such as those in L5 and D29 (45,46) as well as the replication partitioning system of subcluster A2 RepA phages are fully functional outside their phage contexts, it seems likely that they can be readily exchanged between the two, and comparison of cluster A2 genomes suggests this has likely occurred in their relatively recent evolutionary pasts. The genomes of phages Lokk and BobSwaget exemplify this, as they have 97% ANI and identical gene content, except for the additional RepA homologue found in Lokk (47). We are not aware of other sets of closely related genomes where this is observed, and note that this would likely not occur with the prototype lambda phage in which the integration apparatus is wellintegrated into the overall regulatory circuitry, including dependence on the unlinked cII gene for integrase expression (48).…”

Section: Discussionmentioning

confidence: 89%

Protein-Mediated and RNA-Based Origins of Replication of Extrachromosomal Mycobacterial Prophages

Wetzel

Aull

Zack

et al. 2020

mBio

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Temperate bacteriophages are common and establish lysogens of their bacterial hosts in which the prophage is stably inherited. It is typical for such prophages to be integrated into the bacterial chromosome, but extrachromosomally replicating prophages have been described also, with the best characterized being the Escherichia coli phage P1 system. Among the large collection of sequenced mycobacteriophages, more than half are temperate or predicted to be temperate, most of which code for a tyrosine or serine integrase that promotes site-specific prophage integration. However, within the large group of 621 cluster A temperate phages, ∼20% lack an integration cassette, which is replaced with a parABS partitioning system. A subset of these phages carry genes coding for a RepA-like protein (RepA phages), which we show here is necessary and sufficient for autonomous extrachromosomal replication. The non-RepA phages appear to replicate using an RNA-based system, as a parABS-proximal region expressing a noncoding RNA is required for replication. Both RepA and non-RepA phage-based plasmids replicate at one or two copies per cell, transform both Mycobacterium smegmatis and Mycobacterium tuberculosis, and are compatible with pAL5000-derived oriM and integration-proficient plasmid vectors. Characterization of these phage-based plasmids offers insights into the variability of lysogenic maintenance systems and provides a large suite of plasmids for actinobacterial genetics that vary in stability, copy number, compatibility, and host range. IMPORTANCE Bacteriophages are the most abundant biological entities in the biosphere and are a source of uncharacterized biological mechanisms and genetic tools. Here, we identify segments of phage genomes that are used for stable extrachromosomal replication in the prophage state. Autonomous replication of some of these phages requires a RepA-like protein, although most lack repA and use RNA-based systems for replication initiation. We describe a suite of plasmids based on these prophage replication functions that vary in copy number, stability, host range, and compatibility. These plasmids expand the toolbox available for genetic manipulation of Mycobacterium and other Actinobacteria, including Gordonia terrae.

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Section: Discussionmentioning

confidence: 89%

Protein-Mediated and RNA-Based Origins of Replication of Extrachromosomal Mycobacterial Prophages

Wetzel

Aull

Zack

et al. 2020

mBio

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“…In addition, endolysin Lysin B from phage D29 was included as a known positive control that had previously been demonstrated to have the ability to lyse M. smegmatis MC 2 155 cells from without as a purified protein ( Payne and Hatfull, 2012 ). The final selection of eight bacteriophage endolysins chosen, their clusters and respective gene numbers are listed in Table 1 ( Ford et al, 1998 ; Pope et al, 2014 ; Butela et al, 2017 ; Dedrick et al, 2019 ). The genomic regions of these homologs are distinct with the exception of the two A2 cluster phages, StarStuff and D29.…”

Section: Resultsmentioning

confidence: 99%

PLAN-M; Mycobacteriophage Endolysins Fused to Biodegradable Nanobeads Mitigate Mycobacterial Growth in Liquid and on Surfaces

et al. 2021

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The Mycobacteria are a genus of Actinobacteria that include human pathogens such as Mycobacterium tuberculosis (TB). Active TB disease can spread by airborne transmission to healthcare workers and to their community. The HHMI SEA-PHAGES program has contributed to discovering bacteriophages that are able to infect M. smegmatis MC2 155, a close relative of M. tuberculosis. This collection of diverse Mycobacteriophages is an excellent resource for trialling bacteriophage-sourced enzymes in novel applications. Herein we measured the ability Mycobacteriophage endolysins to lyse their host strain when functionally fused to biodegradable polyhydroxyalkanoate (PHA) nanobeads. PHA nanobeads facilitate both the expression and the application of enzymes to surfaces and have been demonstrated to stabilize a wide array of proteins for practical applications whilst eliminating the challenges of traditional protein purification. We selected two Lysin A and six Lysin B homologs to be functionally fused to the polyhydroxyalkanoate synthase C (PhaC). Expression of these constructs resulted in functional lysins displayed on the surface of PHA nanobeads. The lysins thus directionally displayed on nanobeads lysed up to 79% of the M. smegmatis MC2 155 population using 80 mg/mL of nanobeads in pure culture. In order to determine whether the nanobeads would be effective as a protective layer in PPE we adapted a fabric-based test and observed a maximum of 1 log loss of the cell population after 5 h of exposure on a textile (91% cell lysis). Lysin B enzymes performed better than the Lysin A enzymes as a protective barrier on textiles surface assays. These results suggest that bacterial endolysins are efficient in their action when displayed on PHA nanobeads and can cause significant population mortality in as little as 45 min. Our results provide the proof-of-principle that Mycobacteriophage endolysins can be used on functionalized nanobeads where they can protect surfaces such as personal protective equipment (PPE) that routinely come into contact with aerosolised bacteria.

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“…The bacteriophages of mycobacteria have been deeply sampled as a result of the intensive discovery and sequencing efforts of the Howard Hughes Medical Institute’s Science Education Alliance–Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program (http://phagesdb.org) (1, 2). Among these is mycobacteriophage StepMih (GenBank accession number MF185733), the first sequenced from New Zealand (3). Here, we report on the discovery, purification, sequencing, and cluster designations of the following nine new mycobacteriophages: Beatrix, Carthage, Daegal, Dulcie, Fancypants, Fenn, Inca, Naira, and Robyn.…”

Section: Announcementmentioning

confidence: 99%

Complete Genome Sequences of Nine Mycobacteriophages from New Zealand, Beatrix, Carthage, Daegal, Dulcie, Fancypants, Fenn, Inca, Naira, and Robyn

Davies

Wojtus

Gilligan

et al. 2019

Microbiol Resour Announc

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Complete Genome Sequences of Cluster A Mycobacteriophages BobSwaget, Fred313, KADY, Lokk, MyraDee, Stagni, and StepMih

Cited by 4 publications

References 22 publications

Protein-Mediated and RNA-Based Origins of Replication of Extrachromosomal Mycobacterial Prophages

Protein-Mediated and RNA-Based Origins of Replication of Extrachromosomal Mycobacterial Prophages

PLAN-M; Mycobacteriophage Endolysins Fused to Biodegradable Nanobeads Mitigate Mycobacterial Growth in Liquid and on Surfaces

Complete Genome Sequences of Nine Mycobacteriophages from New Zealand, Beatrix, Carthage, Daegal, Dulcie, Fancypants, Fenn, Inca, Naira, and Robyn

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