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

ABSTRACT 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 bes...

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Autores principales:	Katherine S. Wetzel, Haley G. Aull, Kira M. Zack, Rebecca A. Garlena, Graham F. Hatfull
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	Mycobacterium bacteriophage genetics bacteriophages Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/fbe2cda35d5a4f97999fe98da6ec084a
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spelling	oai:doaj.org-article:fbe2cda35d5a4f97999fe98da6ec084a2021-11-15T15:57:03ZProtein-Mediated and RNA-Based Origins of Replication of Extrachromosomal Mycobacterial Prophages10.1128/mBio.00385-202150-7511https://doaj.org/article/fbe2cda35d5a4f97999fe98da6ec084a2020-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00385-20https://doaj.org/toc/2150-7511ABSTRACT 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.Katherine S. WetzelHaley G. AullKira M. ZackRebecca A. GarlenaGraham F. HatfullAmerican Society for MicrobiologyarticleMycobacteriumbacteriophage geneticsbacteriophagesMicrobiologyQR1-502ENmBio, Vol 11, Iss 2 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Mycobacterium bacteriophage genetics bacteriophages Microbiology QR1-502
spellingShingle	Mycobacterium bacteriophage genetics bacteriophages Microbiology QR1-502 Katherine S. Wetzel Haley G. Aull Kira M. Zack Rebecca A. Garlena Graham F. Hatfull Protein-Mediated and RNA-Based Origins of Replication of Extrachromosomal Mycobacterial Prophages
description	ABSTRACT 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.
format	article
author	Katherine S. Wetzel Haley G. Aull Kira M. Zack Rebecca A. Garlena Graham F. Hatfull
author_facet	Katherine S. Wetzel Haley G. Aull Kira M. Zack Rebecca A. Garlena Graham F. Hatfull
author_sort	Katherine S. Wetzel
title	Protein-Mediated and RNA-Based Origins of Replication of Extrachromosomal Mycobacterial Prophages
title_short	Protein-Mediated and RNA-Based Origins of Replication of Extrachromosomal Mycobacterial Prophages
title_full	Protein-Mediated and RNA-Based Origins of Replication of Extrachromosomal Mycobacterial Prophages
title_fullStr	Protein-Mediated and RNA-Based Origins of Replication of Extrachromosomal Mycobacterial Prophages
title_full_unstemmed	Protein-Mediated and RNA-Based Origins of Replication of Extrachromosomal Mycobacterial Prophages
title_sort	protein-mediated and rna-based origins of replication of extrachromosomal mycobacterial prophages
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/fbe2cda35d5a4f97999fe98da6ec084a
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Protein-Mediated and RNA-Based Origins of Replication of Extrachromosomal Mycobacterial Prophages

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