Sequencing, Assembling, and Finishing Complete Bacteriophage Genomes

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.

25A diverse set of prophage-mediated mechanisms protecting bacterial hosts from 26 infection has been recently uncovered within Cluster N mycobacteriophages. In that 27 context, we unveil a novel defense mechanism in Cluster N prophage Butters. By using 28 bioinformatics analyses, phage plating efficiency experiments, microscopy, and 29 immunoprecipitation assays, we show that Butters genes located in the central region of 30 the genome play a key role in the defense against heterotypic viral attack. Our study 31 suggests that a two component system articulated by interactions between protein 32 products of genes 30 and 31 confers defense against heterotypic phage infection by 33 PurpleHaze or Alma, but is insufficient to confer defense against attack by the 34 heterotypic phage Island3. Therefore, based on heterotypic phage plating efficiencies 35 on the Butters lysogen, additional prophage genes required for defense are implicated. 36 37 IMPORTANCE 38 Many sequenced bacterial genomes including pathogenic bacteria contain prophages. 39 Some prophages encode defense systems that protect their bacterial host against 40 heterotypic viral attack. Understanding the mechanisms undergirding these defense 41 systems will be critical to development of phage therapy that circumvents these 42 defenses. Additionally, such knowledge will help engineer phage-resistant bacteria of 43 industrial importance.44 45 47they are useful in diagnostics of mycobacterial infections (1), the most notable of which 48 is tuberculosis (TB), and additionally can serve as genetic tools for mycobacteria (2-5). 49Most recently, engineered mycobacteriophages have been used in therapeutic 50 3 applications to combat infections from antibiotic-resistant strains of Mycobacterium 51 abscessus (6). To date over 11,000 mycobacteriophages have been isolated, over 52 1,800 sequenced, and over 1,600 are available in GenBank (7, 8). Mycobacteriophages 53 are a small subset of the estimated 10 31 bacteriophages existing in the biosphere (9). 54 Mycobacteriophages display high levels of genetic diversity and have been divided into 55 29 genomically similar clusters (A-AC) and a group of singletons with no close relatives 56 (10, 7). Although an increase in isolation and genomic characterization of 57 mycobacteriophages has occurred recently, the void in knowledge about gene 58 expression and function of mycobacteriophage gene products remains. 59 Prophages make up a majority of the known bacteriophage population (11). The 60 relationship between prophages and bacterial strains has shown numerous benefits to 61 both the hosts and phages. Prophages confer many advantages to the host upon 62 integration such as enhanced fitness, reduction of mutation rates, selective advantages, 63 and defense against additional viral attack (12). The bacterial host in turn provides the 64 phages with protection from harsh environments (12). In this context, numerous 65 mechanisms of defense have been recently discovered for Pseudomonas, 66 Mycobacterium, and Gordonia prophages...

One Sentence Summary:Currently data regarding Human Cytomegalovirus resistant mutations are contained in unconnected literature sources, here we present an exhaustive open source database and analysis tool for the community. Abstract:The prevention and treatment of HCMV infection is based on the utilization of antiviral therapies as HCMV lacks an effective vaccine. The rise of drug resistance is therefore an increasing patient threat. We identified the need for an open source and comprehensive HCMV resistance mutations database, to support the research community in this area. Here we present "Cytomegalovirus Drug Resistance Genotyping" (cmvdrg), a freely available database contained within an easily accessible R package, which provides a succinct extraction of literature material in the form of a text file database. Additionally, cmvdrg includes methods for calling resistance in common sequencing files and an optional user-friendly web interface.Availability The cmvdrg package is freely available under the GNU GPL v3 license at https://github.com/ucl-pathgenomics/cmvdrg , Main text: