Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
mBio. 2020 Mar 24;11(2):e00385-20. doi: 10.1128/mBio.00385-20.
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 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 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 -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 and , and are compatible with pAL5000-derived 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. 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 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 and other , including .
温和噬菌体很常见，它们在细菌宿主中建立溶原菌，其中前噬菌体稳定遗传。这种前噬菌体通常整合到细菌染色体中，但也有描述过染色体外复制的前噬菌体，其中最具代表性的是噬菌体 P1 系统。在已测序的分枝杆菌噬菌体中，超过一半是温和噬菌体或预测为温和噬菌体，其中大多数编码酪氨酸或丝氨酸整合酶，促进噬菌体的特异性整合。然而，在 621 个 A 群温和噬菌体中，约有 20%缺乏整合盒，取而代之的是分配系统。这些噬菌体中的一部分携带编码 RepA 样蛋白的基因（RepA 噬菌体），我们在这里证明它是自主染色体外复制所必需和充分的。非 RepA 噬菌体似乎使用基于 RNA 的系统进行复制，因为需要靠近 -近端区域表达非编码 RNA 才能进行复制。RepA 和非 RepA 噬菌体的质粒以每个细胞一到两个拷贝的速度复制，转化 both 和 ，并且与 pAL5000 衍生的 和整合有效质粒载体兼容。这些噬菌体质粒的特性为溶原性维持系统的可变性提供了深入的了解，并提供了一套用于放线菌遗传学的大型质粒，这些质粒在稳定性、拷贝数、兼容性和宿主范围上各不相同。噬菌体是生物圈中最丰富的生物实体，是未被描述的生物机制和遗传工具的来源。在这里，我们确定了噬菌体基因组中用于前噬菌体状态下稳定染色体外复制的片段。这些噬菌体中的一些自主复制需要 RepA 样蛋白，尽管大多数缺乏 RepA 并使用 RNA 为基础的系统进行复制起始。我们描述了一套基于这些噬菌体复制功能的质粒，这些质粒在拷贝数、稳定性、宿主范围和兼容性上各不相同。这些质粒扩展了用于遗传操作的工具包 和其他 ，包括 。
