Holins are bacteriophage-encoded transmembrane proteins that function to control the timing of bacterial lysis event, assist with the destabilization of the membrane proton motive force and in some ...models, generate large “pores” in the cell membrane to allow the exit of the phage-encoded endolysin so they can access the peptidoglycan components of the cell wall. The lysis mechanism has been rigorously evaluated through biochemical and genetic studies in very few phages, and the results indicate that phages utilize endolysins, holins and accessory proteins to the outer membrane to achieve cell lysis through several distinct operational models. This observation suggests the possibility that phages may evolve novel variations of how the lysis proteins functionally interact in an effort to improve fitness or evade host defenses. To begin to address this hypothesis, the current study utilized a comprehensive bioinformatic approach to systematically identify the proteins encoded by the genes within the lysis cassettes in 16 genetically diverse phages that infect the Gram-positive
Gordonia rubripertincta
NRLL B-16540 strain. The results show that there is a high level of diversity of the various lysis genes and 16 different genome organizations of the putative lysis cassette, many which have never been described. Thirty-four different genes encoding holin-like proteins were identified as well as a potential holin-major capsid fusion protein. The holin-like proteins contained between 1–4 transmembrane helices, were not shared to a high degree amongst the different phages and are present in the lysis cassette in a wide range of combinations of up to 4 genes in which none are duplicated. Detailed evaluation of the transmembrane domains and predicted membrane topologies of the holin-like proteins show that many have novel structures that have not been previously characterized. These results provide compelling support that there are novel operational lysis models yet to be discovered.
The bacteriophage population is large, dynamic, ancient, and genetically diverse. Limited genomic information shows that phage genomes are mosaic, and the genetic architecture of phage populations ...remains ill-defined. To understand the population structure of phages infecting a single host strain, we isolated, sequenced, and compared 627 phages of Mycobacterium smegmatis. Their genetic diversity is considerable, and there are 28 distinct genomic types (clusters) with related nucleotide sequences. However, amino acid sequence comparisons show pervasive genomic mosaicism, and quantification of inter-cluster and intra-cluster relatedness reveals a continuum of genetic diversity, albeit with uneven representation of different phages. Furthermore, rarefaction analysis shows that the mycobacteriophage population is not closed, and there is a constant influx of genes from other sources. Phage isolation and analysis was performed by a large consortium of academic institutions, illustrating the substantial benefits of a disseminated, structured program involving large numbers of freshman undergraduates in scientific discovery.
Bacteriophages are the most abundant and diverse entities in the biosphere, and this diversity is driven by constant predator-prey evolutionary dynamics and horizontal gene transfer. Phage genome ...sequences are under-sampled and therefore present an untapped and uncharacterized source of genetic diversity, typically characterized by highly mosaic genomes and no universal genes. To better understand the diversity and relationships among phages infecting human pathogens, we have analysed the complete genome sequences of 205 phages of Staphylococcus sp.
These are predicted to encode 20,579 proteins, which can be sorted into 2139 phamilies (phams) of related sequences; 745 of these are orphams and possess only a single gene. Based on shared gene content, these phages were grouped into four clusters (A, B, C and D), 27 subclusters (A1-A2, B1-B17, C1-C6 and D1-D2) and one singleton. However, the genomes have mosaic architectures and individual genes with common ancestors are positioned in distinct genomic contexts in different clusters. The staphylococcal Cluster B siphoviridae are predicted to be temperate, and the integration cassettes are often closely-linked to genes implicated in bacterial virulence determinants. There are four unusual endolysin organization strategies found in Staphylococcus phage genomes, with endolysins predicted to be encoded as single genes, two genes spliced, two genes adjacent and as a single gene with inter-lytic-domain secondary translational start site. Comparison of the endolysins reveals multi-domain modularity, with conservation of the SH3 cell wall binding domain.
This study provides a high-resolution view of staphylococcal viral genetic diversity, and insights into their gene flux patterns within and across different phage groups (cluster and subclusters) providing insights into their evolution.
Abstract The complete genome sequences of over 220 mycobacteriophages reveal them to be highly diverse, with numerous types sharing little or no nucleotide sequence identity with each other. We have ...determined the preferences of these phages for Mycobacterium tuberculosis and for other strains of Mycobacterium smegmatis , and find there is a correlation between genome type (cluster, subcluster, singleton) and host range. For many of the phages, expansion of host range occurs at relatively high frequencies, and we describe several examples in which host constraints occur at early stages of infection (adsorption or DNA injection), and phages have the ability to expand their host range through mutations in tail genes. We present a model in which phage diversity is a function of both the ability of phages to rapidly adapt to new hosts and the richness of the diversity of the bacterial population from which those phages are isolated.
Large icosahedral viruses that infect bacteria represent an extreme of the coevolution of capsids and the genomes they accommodate. One subset of these large viruses is the jumbophages, tailed phages ...with double-stranded DNA genomes of at least 200,000 bp. We explored the mechanism leading to increased capsid and genome sizes by characterizing structures of several jumbophage capsids and the DNA packaged within them. Capsid structures determined for six jumbophages were consistent with the canonical phage HK97 fold, and three had capsid geometries with novel triangulation numbers (T=25, T=28, and T=52). Packaged DNA (chromosome) sizes were larger than the genome sizes, indicating that all jumbophages use a head-full DNA packaging mechanism. For two phages (PAU and G), the sizes appeared very much larger than their genome length. We used two-dimensional DNA gel electrophoresis to show that these two DNAs migrated abnormally due to base modifications and to allow us to calculate their actual chromosome sizes. Our results support a ratchet model of capsid and genome coevolution whereby mutations lead to increased capsid volume and allow the acquisition of additional genes. Once the added genes and larger capsid are established, mutations that restore the smaller size are disfavored.
A large family of viruses share the same fold of the capsid protein as bacteriophage HK97, a virus that infects bacteria. Members of this family use different numbers of the capsid protein to build capsids of different sizes. Here, we examined the structures of extremely large capsids and measured their DNA content relative to the sequenced genome lengths, aiming to understand the process that increases size. We concluded that mutational changes leading to larger capsids become locked in by subsequent changes to the genome organization.
The global bacteriophage population is large, dynamic, old, and highly diverse genetically. Many phages are tailed and contain double-stranded DNA, but these remain poorly characterized genomically. ...A collection of over 1,000 phages infecting
reveals the diversity of phages of a common bacterial host, but their relationships to phages of phylogenetically proximal hosts are not known. Comparative sequence analysis of 79 phages isolated on
shows these also to be diverse and that the phages can be grouped into 14 clusters of related genomes, with an additional 14 phages that are "singletons" with no closely related genomes. One group of six phages is closely related to Cluster A mycobacteriophages, but the other
phages are distant relatives and share only 10% of their genes with the mycobacteriophages. The
phage genomes vary in genome length (17.1 to 103.4 kb), percentage of GC content (47 to 68.8%), and genome architecture and contain a variety of features not seen in other phage genomes. Like the mycobacteriophages, the highly mosaic
phages demonstrate a spectrum of genetic relationships. We show this is a general property of bacteriophages and suggest that any barriers to genetic exchange are soft and readily violable.
Despite the numerical dominance of bacteriophages in the biosphere, there is a dearth of complete genomic sequences. Current genomic information reveals that phages are highly diverse genomically and have mosaic architectures formed by extensive horizontal genetic exchange. Comparative analysis of 79 phages of
shows them to not only be highly diverse, but to present a spectrum of relatedness. Most are distantly related to phages of the phylogenetically proximal host
, although one group of
phages is more closely related to mycobacteriophages than to the other
phages. Phage genome sequence space remains largely unexplored, but further isolation and genomic comparison of phages targeted at related groups of hosts promise to reveal pathways of bacteriophage evolution.
Purpose
To compare titer reduction and delivery rate of active anti-tuberculosis bacteriophage (phage) D29 with three inhalation devices.
Methods
Phage D29 lysate was amplified to a titer of ...11.8 ± 0.3 log
10
(pfu/mL) and diluted 1:100 in isotonic saline. Filters captured the aerosolized saline D29 preparation emitted from three types of inhalation devices: 1) vibrating mesh nebulizer; 2) jet nebulizer; 3) soft mist inhaler. Full-plate plaque assays, performed in triplicate at multiple dilution levels with the surrogate host
Mycobacterium smegmatis
, were used to quantify phage titer
.
Results
Respective titer reductions for the vibrating mesh nebulizer, jet nebulizer, and soft mist inhaler were 0.4 ± 0.1, 3.7 ± 0.1, and 0.6 ± 0.3 log
10
(pfu/mL). Active phage delivery rate was significantly greater (
p
< 0.01) for the vibrating mesh nebulizer (3.3x10
8
± 0.8x10
8
pfu/min) than for the jet nebulizer (5.4x10
4
± 1.3x10
4
pfu/min). The soft mist inhaler delivered 4.6x10
6
± 2.0x10
6
pfu per 11.6 ± 1.6 μL ex-actuator dose.
Conclusions
Delivering active phage requires a prudent choice of inhalation device. The jet nebulizer was not a good choice for aerosolizing phage D29 under the tested conditions, due to substantial titer reduction likely occurring during droplet production. The vibrating mesh nebulizer is recommended for animal inhalation studies requiring large amounts of D29 aerosol, whereas the soft mist inhaler may be useful for self-administration of D29 aerosol.
Engaging large numbers of undergraduates in authentic scientific discovery is desirable but difficult to achieve. We have developed a general model in which faculty and teaching assistants from ...diverse academic institutions are trained to teach a research course for first-year undergraduate students focused on bacteriophage discovery and genomics. The course is situated within a broader scientific context aimed at understanding viral diversity, such that faculty and students are collaborators with established researchers in the field. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) course has been widely implemented and has been taken by over 4,800 students at 73 institutions. We show here that this alliance-sourced model not only substantially advances the field of phage genomics but also stimulates students' interest in science, positively influences academic achievement, and enhances persistence in science, technology, engineering, and mathematics (STEM) disciplines. Broad application of this model by integrating other research areas with large numbers of early-career undergraduate students has the potential to be transformative in science education and research training.
Engagement of undergraduate students in scientific research at early stages in their careers presents an opportunity to excite students about science, technology, engineering, and mathematics (STEM) disciplines and promote continued interests in these areas. Many excellent course-based undergraduate research experiences have been developed, but scaling these to a broader impact with larger numbers of students is challenging. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunting Advancing Genomics and Evolutionary Science (SEA-PHAGES) program takes advantage of the huge size and diversity of the bacteriophage population to engage students in discovery of new viruses, genome annotation, and comparative genomics, with strong impacts on bacteriophage research, increased persistence in STEM fields, and student self-identification with learning gains, motivation, attitude, and career aspirations.
Glycosylation of eukaryotic virus particles is common and influences their uptake, trafficking, and immune recognition. In contrast, glycosylation of bacteriophage particles has not been reported; ...phage virions typically do not enter the cytoplasm upon infection, and they do not generally inhabit eukaryotic systems. We show here that several genomically distinct phages of Mycobacteria are modified with glycans attached to the C terminus of capsid and tail tube protein subunits. These O-linked glycans influence antibody production and recognition, shielding viral particles from antibody binding and reducing production of neutralizing antibodies. Glycosylation is mediated by phage-encoded glycosyltransferases, and genomic analysis suggests that they are relatively common among mycobacteriophages. Putative glycosyltransferases are also encoded by some Gordonia and Streptomyces phages, but there is little evidence of glycosylation among the broader phage population. The immune response to glycosylated phage virions in mice suggests that glycosylation may be an advantageous property for phage therapy of Mycobacterium infections.
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•Some mycobacteriophages are glycosylated on capsid and/or tail tube subunits•Glycosylation is mediated by phage-encoded glycosyltransferases•The glycans are large and complex and vary significantly between phages•Glycans alter antibody production and shield viral particles from antibody binding
Freeman et al. describe mycobacteriophages with glycosylated capsid and/or tail tube protein subunits. This modification is mediated by phage-encoded glycosyltransferases, and there is evidence for glycosylation throughout the mycobacteriophage population. Glycans alter the immune processing of phages and the binding affinity of antibodies to intact phages.
Mycobacteriophages are viruses that infect mycobacterial hosts. Expansion of a collection of sequenced phage genomes to a total of 60—all infecting a common bacterial host—provides further insight ...into their diversity and evolution. Of the 60 phage genomes, 55 can be grouped into nine clusters according to their nucleotide sequence similarities, 5 of which can be further divided into subclusters; 5 genomes do not cluster with other phages. The sequence diversity between genomes within a cluster varies greatly; for example, the 6 genomes in Cluster D share more than 97.5% average nucleotide similarity with one another. In contrast, similarity between the 2 genomes in Cluster I is barely detectable by diagonal plot analysis. In total, 6858 predicted open-reading frames have been grouped into 1523 phamilies (phams) of related sequences, 46% of which possess only a single member. Only 18.8% of the phams have sequence similarity to non-mycobacteriophage database entries, and fewer than 10% of all phams can be assigned functions based on database searching or synteny. Genome clustering facilitates the identification of genes that are in greatest genetic flux and are more likely to have been exchanged horizontally in relatively recent evolutionary time. Although mycobacteriophage genes exhibit a smaller average size than genes of their host (205 residues compared with 315), phage genes in higher flux average only 100 amino acids, suggesting that the primary units of genetic exchange correspond to single protein domains.
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