DEPhT: a novel approach for efficient prophage discovery and precise extraction.

Advances in genome sequencing have produced hundreds of thousands of bacterial genome sequences, many of which have integrated prophages derived from temperate bacteriophages. These prophages play key roles by influencing bacterial metabolism, pathogenicity, antibiotic resistance, and defense against viral attack. However, they vary considerably even among related bacterial strains, and they are challenging to identify computationally and to extract precisely for comparative genomic analyses. Here, we describe DEPhT, a multimodal tool for prophage discovery and extraction. It has three run modes that facilitate rapid screening of large numbers of bacterial genomes, precise extraction of prophage sequences, and prophage annotation. DEPhT uses genomic architectural features that discriminate between phage and bacterial sequences for efficient prophage discovery, and targeted homology searches for precise prophage extraction. DEPhT is designed for prophage discovery in Mycobacterium genomes but can be adapted broadly to other bacteria. We deploy DEPhT to demonstrate that prophages are prevalent in Mycobacterium strains but are absent not only from the few well-characterized Mycobacterium tuberculosis strains, but also are absent from all ∼30 000 sequenced M. tuberculosis strains.

Introduction to the special issue on education in acoustics.

A substantial fraction of the membership of the Acoustical Society of America are faculty at various types of educational institutions and are actively engaged in educational activities. However, papers focusing on aspects of teaching, pedagogy, demonstrations, student learning, and other education topics are not often published in JASA, even though the Education in Acoustics Committee regularly offers special sessions on these topics at every ASA meeting. This special issue of JASA dedicated to Education in Acoustics includes 41 papers from authors all over the world. This introduction to the special issue briefly describes each of the papers, which have been organized into several broad categories: teaching methods and exercises; project-based learning; use of experiments, demos, and experiential learning; adapting to teaching during COVID-19; circuit models and impedance concepts; software apps and online resources; teaching musical acoustics; and descriptions of acoustics programs at a variety of institutions.

An inclusive Research Education Community (iREC): Impact of the SEA-PHAGES program on research outcomes and student learning.

Engaging undergraduate students in scientific research promises substantial benefits, but it is not accessible to all students and is rarely implemented early in college education, when it will have the greatest impact. An inclusive Research Education Community (iREC) provides a centralized scientific and administrative infrastructure enabling engagement of large numbers of students at different types of institutions. The Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) is an iREC that promotes engagement and continued involvement in science among beginning undergraduate students. The SEA-PHAGES students show strong gains correlated with persistence relative to those in traditional laboratory courses regardless of academic, ethnic, gender, and socioeconomic profiles. This persistent involvement in science is reflected in key measures, including project ownership, scientific community values, science identity, and scientific networking.

PhagesDB: the actinobacteriophage database.

The Actinobacteriophage Database (PhagesDB) is a comprehensive, interactive, database-backed website that collects and shares information related to the discovery, characterization and genomics of viruses that infect Actinobacterial hosts. To date, more than 8000 bacteriophages-including over 1600 with sequenced genomes-have been entered into the database. PhagesDB plays a crucial role in organizing the discoveries of phage biologists around the world-including students in the SEA-PHAGES program-and has been cited in over 50 peer-reviewed articles. Availability and Implementation: http://phagesdb.org/. Contact: gfh@pitt.edu.

Complete genomic sequences of Propionibacterium freudenreichii phages from Swiss cheese reveal greater diversity than Cutibacterium (formerly Propionibacterium) acnes phages.

BACKGROUND: A remarkable exception to the large genetic diversity often observed for bacteriophages infecting a specific bacterial host was found for the Cutibacterium acnes (formerly Propionibacterium acnes) phages, which are highly homogeneous. Phages infecting the related species, which is also a member of the Propionibacteriaceae family, Propionibacterium freudenreichii, a bacterium used in production of Swiss-type cheeses, have also been described and are common contaminants of the cheese manufacturing process. However, little is known about their genetic composition and diversity. RESULTS: We obtained seven independently isolated bacteriophages that infect P. freudenreichii from Swiss-type cheese samples, and determined their complete genome sequences. These data revealed that all seven phage isolates are of similar genomic length and GC% content, but their genomes are highly diverse, including genes encoding the capsid, tape measure, and tail proteins. In contrast to C. acnes phages, all P. freudenreichii phage genomes encode a putative integrase protein, suggesting they are capable of lysogenic growth. This is supported by the finding of related prophages in some P. freudenreichii strains. The seven phages could further be distinguished as belonging to two distinct genomic types, or 'clusters', based on nucleotide sequences, and host range analyses conducted on a collection of P. freudenreichii strains show a higher degree of host specificity than is observed for the C. acnes phages. CONCLUSIONS: Overall, our data demonstrate P. freudenreichii bacteriophages are distinct from C. acnes phages, as evidenced by their higher genetic diversity, potential for lysogenic growth, and more restricted host ranges. This suggests substantial differences in the evolution of these related species from the Propionibacteriaceae family and their phages, which is potentially related to their distinct environmental niches.

Acoustics of ping-pong: Vibroacoustic analysis of table tennis rackets and balls.

The sound resulting from the impact of a table tennis racket and ball can influence a player's perception of equipment quality in addition to providing clues to personal performance. This study explores the vibrational modes of both racket and ball and how those modes contribute to the impact sound. Experimental modal analysis reveals that the racket exhibits a large number of structural vibration modes typical of elliptical plates. Acoustic analysis reveals that two of those structural modes dominate the sound produced by the ball-paddle impact. The rubber padding provides some damping and a significant mass loading to the racket vibrations. The hollow cellulose nitrate balls exhibit vibrational modes typical of a hollow spherical shell, starting with frequencies around 5920 Hz. Experimental frequencies confirm theoretical and computational models. However, the contact time between racket and ball is long enough that the lowest acoustic modes of the ball do not contribute to the radiated sound. Instead, acoustic analysis suggests that the ball appears to radiate sound at a much higher frequency sound (8.5-12 kHz) most likely due to snap-through after buckling common to spherical shells undergoing deformation while impacting a rigid surface at high speeds.

Function, expression, specificity, diversity and incompatibility of actinobacteriophage parABS systems.

More than 180 individual phages infecting hosts in the phylum Actinobacteria have been sequenced and grouped into Cluster A because of their similar overall nucleotide sequences and genome architectures. These Cluster A phages are either temperate or derivatives of temperate parents, and most have an integration cassette near the centre of the genome containing an integrase gene and attP. However, about 20% of the phages lack an integration cassette, which is replaced by a 1.4 kbp segment with predicted partitioning functions, including plasmid-like parA and parB genes. Phage RedRock forms stable lysogens in Mycobacterium smegmatis in which the prophage replicates at 2.4 copies/chromosome and the partitioning system confers prophage maintenance. The parAB genes are expressed upon RedRock infection of M. smegmatis, but are downregulated once lysogeny is established by binding of RedRock ParB to parS-L, one of two centromere-like sites flanking the parAB genes. The RedRock parS-L and parS-R sites are composed of eight directly repeated copies of an 8 bp motif that is recognized by ParB. The actinobacteriophage parABS cassettes span considerable sequence diversity and specificity, providing a suite of tools for use in mycobacterial genetics.

Characterization of mycobacteria and mycobacteriophages isolated from compost at the São Paulo Zoo Park Foundation in Brazil and creation of the new mycobacteriophage Cluster U.

BACKGROUND: A large collection of sequenced mycobacteriophages capable of infecting a single host strain of Mycobacterium smegmatis shows considerable genomic diversity with dozens of distinctive types (clusters) and extensive variation within those sharing evident nucleotide sequence similarity. Here we profiled the mycobacterial components of a large composting system at the São Paulo zoo. RESULTS: We isolated and sequenced eight mycobacteriophages using Mycobacterium smegmatis mc(2)155 as a host. None of these eight phages infected any of mycobacterial strains isolated from the same materials. The phage isolates span considerable genomic diversity, including two phages (Barriga, Nhonho) related to Subcluster A1 phages, two Cluster B phages (Pops, Subcluster B1; Godines, Subcluster B2), three Subcluster F1 phages (Florinda, Girafales, and Quico), and Madruga, a relative of phage Patience with which it constitutes the new Cluster U. Interestingly, the two Subcluster A1 phages and the three Subcluster F1 phages have genomic relationships indicating relatively recent evolution within a geographically isolated niche in the composting system. CONCLUSIONS: We predict that composting systems such as those used to obtain these mycobacteriophages will be a rich source for the isolation of additional phages that will expand our view of bacteriophage diversity and evolution.

Structure of the archaeal head-tailed virus HSTV-1 completes the HK97 fold story.

It has been proposed that viruses can be divided into a small number of structure-based viral lineages. One of these lineages is exemplified by bacterial virus Hong Kong 97 (HK97), which represents the head-tailed dsDNA bacteriophages. Seemingly similar viruses also infect archaea. Here we demonstrate using genomic analysis, electron cryomicroscopy, and image reconstruction that the major coat protein fold of newly isolated archaeal Haloarcula sinaiiensis tailed virus 1 has the canonical coat protein fold of HK97. Although it has been anticipated previously, this is physical evidence that bacterial and archaeal head-tailed viruses share a common architectural principle. The HK97-like fold has previously been recognized also in herpesviruses, and this study expands the HK97-like lineage to viruses from all three domains of life. This is only the second established lineage to include archaeal, bacterial, and eukaryotic viruses. Thus, our findings support the hypothesis that the last common universal ancestor of cellular organisms was infected by a number of different viruses.

Cluster M mycobacteriophages Bongo, PegLeg, and Rey with unusually large repertoires of tRNA isotypes.

UNLABELLED: Genomic analysis of a large set of phages infecting the common host Mycobacterium smegmatis mc(2)155 shows that they span considerable genetic diversity. There are more than 20 distinct types that lack nucleotide similarity with each other, and there is considerable diversity within most of the groups. Three newly isolated temperate mycobacteriophages, Bongo, PegLeg, and Rey, constitute a new group (cluster M), with the closely related phages Bongo and PegLeg forming subcluster M1 and the more distantly related Rey forming subcluster M2. The cluster M mycobacteriophages have siphoviral morphologies with unusually long tails, are homoimmune, and have larger than average genomes (80.2 to 83.7 kbp). They exhibit a variety of features not previously described in other mycobacteriophages, including noncanonical genome architectures and several unusual sets of conserved repeated sequences suggesting novel regulatory systems for both transcription and translation. In addition to containing transfer-messenger RNA and RtcB-like RNA ligase genes, their genomes encode 21 to 24 tRNA genes encompassing complete or nearly complete sets of isotypes. We predict that these tRNAs are used in late lytic growth, likely compensating for the degradation or inadequacy of host tRNAs. They may represent a complete set of tRNAs necessary for late lytic growth, especially when taken together with the apparent lack of codons in the same late genes that correspond to tRNAs that the genomes of the phages do not obviously encode. IMPORTANCE: The bacteriophage population is vast, dynamic, and old and plays a central role in bacterial pathogenicity. We know surprisingly little about the genetic diversity of the phage population, although metagenomic and phage genome sequencing indicates that it is great. Probing the depth of genetic diversity of phages of a common host, Mycobacterium smegmatis, provides a higher resolution of the phage population and how it has evolved. Three new phages constituting a new cluster M further expand the diversity of the mycobacteriophages and introduce novel features. As such, they provide insights into phage genome architecture, virion structure, and gene regulation at the transcriptional and translational levels.

Insights into head-tailed viruses infecting extremely halophilic archaea.

Extremophilic archaea, both hyperthermophiles and halophiles, dominate in habitats where rather harsh conditions are encountered. Like all other organisms, archaeal cells are susceptible to viral infections, and to date, about 100 archaeal viruses have been described. Among them, there are extraordinary virion morphologies as well as the common head-tailed viruses. Although approximately half of the isolated archaeal viruses belong to the latter group, no three-dimensional virion structures of these head-tailed viruses are available. Thus, rigorous comparisons with bacteriophages are not yet warranted. In the present study, we determined the genome sequences of two of such viruses of halophiles and solved their capsid structures by cryo-electron microscopy and three-dimensional image reconstruction. We show that these viruses are inactivated, yet remain intact, at low salinity and that their infectivity is regained when high salinity is restored. This enabled us to determine their three-dimensional capsid structures at low salinity to a ∼10-Šresolution. The genetic and structural data showed that both viruses belong to the same T-number class, but one of them has enlarged its capsid to accommodate a larger genome than typically associated with a T=7 capsid by inserting an additional protein into the capsid lattice.

Genome sequences of 31 mycobacteriophages isolated on Mycobacterium smegmatis mc2155 at room temperature.

We report the genome sequences of 31 mycobacteriophages isolated on Mycobacterium smegmatis mc2155 at room temperature. The genomes add to the diversity of Clusters A, B, C, G, and K. Collectively, the genomes include 70 novel protein-coding genes that have no close relatives among the actinobacteriophages.

Snapshot of haloarchaeal tailed virus genomes.

The complete genome sequences of archaeal tailed viruses are currently highly underrepresented in sequence databases. Here, we report the genomic sequences of 10 new tailed viruses infecting different haloarchaeal hosts. Among these, only two viral genomes are closely related to each other and to previously described haloviruses HF1 and HF2. The approximately 760 kb of new genomic sequences in total shows no matches to CRISPR/Cas spacer sequences in haloarchaeal host genomes. Despite their high divergence, we were able to identify virion structural and assembly genes as well as genes coding for DNA and RNA metabolic functions. Interestingly, we identified many genes and genomic features that are shared with tailed bacteriophages, consistent with the hypothesis that haloarchaeal and bacterial tailed viruses share common ancestry, and that a viral lineage containing archaeal viruses, bacteriophages and eukaryotic viruses predates the division of the three major domains of non-viral life. However, as in tailed viruses in general and in haloarchaeal tailed viruses in particular, there are still a considerable number of predicted genes of unknown function.

Unusual prophages in Mycobacterium abscessus genomes and strain variations in phage susceptibilities.

Mycobacterium abscessus infections are relatively common in patients with cystic fibrosis and are clinically challenging, with frequent intrinsic resistance to antibiotics. Therapeutic treatment with bacteriophages offers some promise but faces many challenges including substantial variation in phage susceptibilities among clinical isolates, and the need to personalize therapies for individual patients. Many strains are not susceptible to any phages or are not efficiently killed by lytic phages, including all smooth colony morphotype strains tested to-date. Here, we analyze a set of new M. abscessus isolates for the genomic relationships, prophage content, spontaneous phage release, and phage susceptibilities. We find that prophages are common in these M. abscessus genomes, but some have unusual arrangements, including tandemly integrated prophages, internal duplications, and they participate in active exchange of polymorphic toxin-immunity cassettes secreted by ESX systems. Relatively few strains are efficiently infected by any mycobacteriophages, and the infection patterns do not reflect the overall phylogenetic relationships of the strains. Characterization of these strains and their phage susceptibility profiles will help to advance the broader application of phage therapies for NTM infections.

Therapeutically useful mycobacteriophages BPs and Muddy require trehalose polyphleates.

Mycobacteriophages show promise as therapeutic agents for non-tuberculous mycobacterium infections. However, little is known about phage recognition of Mycobacterium cell surfaces or mechanisms of phage resistance. We show here that trehalose polyphleates (TPPs)-high-molecular-weight, surface-exposed glycolipids found in some mycobacterial species-are required for infection of Mycobacterium abscessus and Mycobacterium smegmatis by clinically useful phages BPs and Muddy. TPP loss leads to defects in adsorption and infection and confers resistance. Transposon mutagenesis shows that TPP disruption is the primary mechanism for phage resistance. Spontaneous phage resistance occurs through TPP loss by mutation, and some M. abscessus clinical isolates are naturally phage-insensitive due to TPP synthesis gene mutations. Both BPs and Muddy become TPP-independent through single amino acid substitutions in their tail spike proteins, and M. abscessus mutants resistant to TPP-independent phages reveal additional resistance mechanisms. Clinical use of BPs and Muddy TPP-independent mutants should preempt phage resistance caused by TPP loss.

Mycobacterium trehalose polyphleates are required for infection by therapeutically useful mycobacteriophages BPs and Muddy.

Mycobacteriophages are good model systems for understanding their bacterial hosts and show promise as therapeutic agents for nontuberculous mycobacterium infections. However, little is known about phage recognition of Mycobacterium cell surfaces, or mechanisms of phage resistance. We show here that surface-exposed trehalose polyphleates (TPPs) are required for infection of Mycobacterium abscessus and Mycobacterium smegmatis by clinically useful phages BPs and Muddy, and that TPP loss leads to defects in adsorption, infection, and confers resistance. Transposon mutagenesis indicates that TPP loss is the primary mechanism for phage resistance. Spontaneous phage resistance occurs through TPP loss, and some M. abscessus clinical isolates are phage-insensitive due to TPP absence. Both BPs and Muddy become TPP-independent through single amino acid substitutions in their tail spike proteins, and M. abscessus mutants resistant to TPP-independent phages reveal additional resistance mechanisms. Clinical use of BPs and Muddy TPP-independent mutants should preempt phage resistance caused by TPP loss.

Mycobacterial nucleoid-associated protein Lsr2 is required for productive mycobacteriophage infection.

Mycobacteriophages are a diverse group of viruses infecting Mycobacterium with substantial therapeutic potential. However, as this potential becomes realized, the molecular details of phage infection and mechanisms of resistance remain ill-defined. Here we use live-cell fluorescence microscopy to visualize the spatiotemporal dynamics of mycobacteriophage infection in single cells and populations, showing that infection is dependent on the host nucleoid-associated Lsr2 protein. Mycobacteriophages preferentially adsorb at Mycobacterium smegmatis sites of new cell wall synthesis and following DNA injection, Lsr2 reorganizes away from host replication foci to establish zones of phage DNA replication (ZOPR). Cells lacking Lsr2 proceed through to cell lysis when infected but fail to generate consecutive phage bursts that trigger epidemic spread of phage particles to neighbouring cells. Many mycobacteriophages code for their own Lsr2-related proteins, and although their roles are unknown, they do not rescue the loss of host Lsr2.

Acoustic testing and modeling: an advanced undergraduate laboratory.

This paper describes an advanced laboratory course in acoustics, specifically targeted for students with an interest in engineering applications at a school with a strongly integrated industrial co-op program. The laboratory course is developed around a three-pronged approach to problem solving that combines and integrates theoretical models, computational models, and experimental data. The course is structured around modules that begin with fundamental concepts and build laboratory skills and expand the knowledge base toward a final project. Students keep a detailed laboratory notebook, write research papers in teams, and must pass laboratory certification exams. This paper describes the course layout and philosophy and shares personal experience from both faculty and student perspectives.

Broadening Access to STEM through the Community College: Investigating the Role of Course-Based Research Experiences (CREs).

Broadening access to science, technology, engineering, and mathematics (STEM) professions through the provision of early-career research experiences for a wide range of demographic groups is important for the diversification of the STEM workforce. The size and diversity of the community college system make it a prime educational site for achieving this aim. However, some evidence shows that women and Black, Latinx, and Native American student groups have been hindered in STEM at the community college level. One option for enhancing persistence in STEM is to incorporate the course-based research experiences (CREs) into the curriculum as a replacement for the prevalent traditional laboratory. This can be achieved through the integration of community colleges within extant, multi-institutional CREs such as the SEA-PHAGES program. Using a propensity score-matching technique, students in a CRE and traditional laboratory were compared on a range of psychosocial variables (project ownership, self-efficacy, science identity, scientific community values, and networking). Results revealed higher ratings for women and persons excluded because of their ethnicity or race (PEERs) in the SEA-PHAGES program on important predictors of persistence such as project ownership and science identity. This suggests that the usage of CREs at community colleges could have positive effects in addressing the gender gap for women and enhance inclusiveness for PEER students in STEM.

Toward a Phage Cocktail for Tuberculosis: Susceptibility and Tuberculocidal Action of Mycobacteriophages against Diverse Mycobacterium tuberculosis Strains.

The global health burden of human tuberculosis (TB) and the widespread antibiotic resistance of its causative agent Mycobacterium tuberculosis warrant new strategies for TB control. The successful use of a bacteriophage cocktail to treat a Mycobacterium abscessus infection suggests that phages could play a role in tuberculosis therapy. To assemble a phage cocktail with optimal therapeutic potential for tuberculosis, we have explored mycobacteriophage diversity to identify phages that demonstrate tuberculocidal activity and determined the phage infection profiles for a diverse set of strains spanning the major lineages of human-adapted strains of the Mycobacterium tuberculosis complex. Using a combination of genome engineering and bacteriophage genetics, we have assembled a five-phage cocktail that minimizes the emergence of phage resistance and cross-resistance to multiple phages, and which efficiently kills the M. tuberculosis strains tested. Furthermore, these phages function without antagonizing antibiotic effectiveness, and infect both isoniazid-resistant and -sensitive strains.IMPORTANCE Tuberculosis kills 1.5 million people each year, and resistance to commonly used antibiotics contributes to treatment failures. The therapeutic potential of bacteriophages against Mycobacterium tuberculosis offers prospects for shortening antibiotic regimens, provides new tools for treating multiple drug-resistant (MDR)-TB and extensively drug-resistant (XDR)-TB infections, and protects newly developed antibiotics against rapidly emerging resistance to them. Identifying a suitable suite of phages active against diverse M. tuberculosis isolates circumvents many of the barriers to initiating clinical evaluation of phages as part of the arsenal of antituberculosis therapeutics.