Targeting intracellular nontuberculous mycobacteria and M. tuberculosis with a bactericidal enzymatic cocktail.

To address intracellular mycobacterial infections, we developed a cocktail of four enzymes that catalytically attack three layers of the mycobacterial envelope. This cocktail is delivered to macrophages, through a targeted liposome presented here as ENTX_001. Endolytix Cocktail 1 (EC1) leverages mycobacteriophage lysin enzymes LysA and LysB, while also including α-amylase and isoamylase for degradation of the mycobacterial envelope from outside of the cell. The LysA family of proteins from mycobacteriophages has been shown to cleave the peptidoglycan layer, whereas LysB is an esterase that hydrolyzes the linkage between arabinogalactan and mycolic acids of the mycomembrane. The challenge of gaining access to the substrates of LysA and LysB provided exogenously was addressed by adding amylase enzymes that degrade the extracellular capsule shown to be present in Mycobacterium tuberculosis. This enzybiotic approach avoids antimicrobial resistance, specific receptor-mediated binding, and intracellular DNA surveillance pathways that limit many bacteriophage applications. We show this cocktail of enzymes is bactericidal in vitro against both rapid- and slow-growing nontuberculous mycobacteria (NTM) as well as M. tuberculosis strains. The EC1 cocktail shows superior killing activity when compared to previously characterized LysB alone. EC1 is also powerfully synergistic with standard-of-care antibiotics. In addition to in vitro killing of NTM, ENTX_001 demonstrates the rescue of infected macrophages from necrotic death by Mycobacteroides abscessus and Mycobacterium avium. Here, we demonstrate shredding of mycobacterial cells by EC1 into cellular debris as a mechanism of bactericide.IMPORTANCEThe world needs entirely new forms of antibiotics as resistance to chemical antibiotics is a critical problem facing society. We addressed this need by developing a targeted enzyme therapy for a broad range of species and strains within mycobacteria and highly related genera including nontuberculous mycobacteria such as Mycobacteroides abscessus, Mycobacterium avium, Mycobacterium intracellulare, as well as Mycobacterium tuberculosis. One advantage of this approach is the ability to drive our lytic enzymes through encapsulation into macrophage-targeted liposomes resulting in attack of mycobacteria in the cells that harbor them where they hide from the adaptive immune system and grow. Furthermore, this approach shreds mycobacteria independent of cell physiology as the drug targets the mycobacterial envelope while sidestepping the host range limitations observed with phage therapy and resistance to chemical antibiotics.

A genome-wide cytotoxicity screen of cluster F1 mycobacteriophage Girr reveals novel inhibitors of Mycobacterium smegmatis growth.

Over the past decade, thousands of bacteriophage genomes have been sequenced and annotated. A striking observation from this work is that known structural features and functions cannot be assigned for >65% of the encoded proteins. One approach to begin experimentally elucidating the function of these uncharacterized gene products is genome-wide screening to identify phage genes that confer phenotypes of interest like inhibition of host growth. This study describes the results of a screen evaluating the effects of overexpressing each gene encoded by the temperate Cluster F1 mycobacteriophage Girr on the growth of the host bacterium Mycobacterium smegmatis. Overexpression of 29 of the 102 Girr genes (~28% of the genome) resulted in mild to severe cytotoxicity. Of the 29 toxic genes described, 12 have no known function and are predominately small proteins of <125 amino acids. Overexpression of the majority of these 12 cytotoxic no known functions proteins resulted in moderate to severe growth reduction and represent novel antimicrobial products. The remaining 17 toxic genes have predicted functions, encoding products involved in phage structure, DNA replication/modification, DNA binding/gene regulation, or other enzymatic activity. Comparison of this dataset with prior genome-wide cytotoxicity screens of mycobacteriophages Waterfoul and Hammy reveals some common functional themes, though several of the predicted Girr functions associated with cytotoxicity in our report, including genes involved in lysogeny, have not been described previously. This study, completed as part of the HHMI-supported SEA-GENES project, highlights the power of parallel, genome-wide overexpression screens to identify novel interactions between phages and their hosts.

A genome-wide overexpression screen reveals Mycobacterium smegmatis growth inhibitors encoded by mycobacteriophage Hammy.

During infection, bacteriophages produce diverse gene products to overcome bacterial antiphage defenses, to outcompete other phages, and to take over cellular processes. Even in the best-studied model phages, the roles of most phage-encoded gene products are unknown, and the phage population represents a largely untapped reservoir of novel gene functions. Considering the sheer size of this population, experimental screening methods are needed to sort through the enormous collection of available sequences and identify gene products that can modulate bacterial behavior for downstream functional characterization. Here, we describe the construction of a plasmid-based overexpression library of 94 genes encoded by Hammy, a Cluster K mycobacteriophage closely related to those infecting clinically important mycobacteria. The arrayed library was systematically screened in a plate-based cytotoxicity assay, identifying a diverse set of 24 gene products (representing ∼25% of the Hammy genome) capable of inhibiting growth of the host bacterium Mycobacterium smegmatis. Half of these are related to growth inhibitors previously identified in related phage Waterfoul, supporting their functional conservation; the other genes represent novel additions to the list of known antimycobacterial growth inhibitors. This work, conducted as part of the HHMI-supported Science Education Alliance Gene-function Exploration by a Network of Emerging Scientists (SEA-GENES) project, highlights the value of parallel, comprehensive overexpression screens in exploring genome-wide patterns of phage gene function and novel interactions between phages and their hosts.

The heterogenous and diverse population of prophages in Mycobacterium genomes.

IMPORTANCE: Mycobacterium species include several human pathogens and mycobacteriophages show potential for therapeutic use to control Mycobacterium infections. However, phage infection profiles vary greatly among Mycobacterium abscessus clinical isolates and phage therapies must be personalized for individual patients. Mycobacterium phage susceptibility is likely determined primarily by accessory parts of bacterial genomes, and we have identified the prophage and phage-related genomic regions across sequenced Mycobacterium strains. The prophages are numerous and diverse, especially in M. abscessus genomes, and provide a potentially rich reservoir of new viruses that can be propagated lytically and used to expand the repertoire of therapeutically useful phages.

Characterization of novel recombinant mycobacteriophages derived from homologous recombination between two temperate phages.

Comparative analyses of mycobacteriophage genomes reveals extensive genetic diversity in genome organization and gene content, contributing to widespread mosaicism. We previously reported that the prophage of mycobacteriophage Butters (cluster N) provides defense against infection by Island3 (subcluster I1). To explore the anti-Island3 defense mechanism, we attempted to isolate Island3 defense escape mutants on a Butters lysogen, but only uncovered phages with recombinant genomes comprised of regions of Butters and Island3 arranged from left arm to right arm as Butters-Island3-Butters (BIBs). Recombination occurs within two distinct homologous regions that encompass lysin A, lysin B, and holin genes in one segment, and RecE and RecT genes in the other. Structural genes of mosaic BIB genomes are contributed by Butters while the immunity cassette is derived from Island3. Consequently, BIBs are morphologically identical to Butters (as shown by transmission electron microscopy) but are homoimmune with Island3. Recombinant phages overcome antiphage defense and silencing of the lytic cycle. We leverage this observation to propose a stratagem to generate novel phages for potential therapeutic use.

Isolation and characterization of a novel mycobacteriophage Kashi-VT1 infecting Mycobacterium species.

Mycobacteriophages are viruses that infect members of genus Mycobacterium. Because of the rise in antibiotic resistance in mycobacterial diseases such as tuberculosis, mycobacteriophages have received renewed attention as alternative therapeutic agents. Mycobacteriophages are highly diverse, and, on the basis of their genome sequences, they are grouped into 30 clusters and 10 singletons. In this article, we have described the isolation and characterization of a novel mycobacteriophage Kashi-VT1 (KVT1) infecting Mycobacterium >smegmatis mc2 155 (M. smegmatis) and Mycobacterium fortuitum isolated from Varanasi, India. KVT1 is a cluster K1 temperate phage that belongs to Siphoviridae family as visualized in transmission electron microscopy. The phage genome is 61,010 base pairs with 66.5% Guanine/Cytosine (GC) content, encoding 101 putative open reading frames. The KVT1 genome encodes an immunity repressor, a tyrosine integrase, and an excise protein, which are the characteristics of temperate phages. It also contains genes encoding holin, lysin A, and lysin B involved in host cell lysis. The one-step growth curve demonstrated that KVT1 has a latency time of 90 min and an average burst size of 101 phage particles per infected cell. It can withstand a temperature of up to 45°C and has a maximum viability between pH 8 and 9. Some mycobacteriophages from cluster K are known to infect the pathogenic Mycobacterium tuberculosis (M. tuberculosis); hence, KVT1 holds potential for the phage therapy against tuberculosis, and it can also be engineered to convert into an exclusively lytic phage.

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.

Virion glycosylation influences mycobacteriophage immune recognition.

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.

Identification of Toxic Proteins Encoded by Mycobacteriophage TM4 Using a Next-Generation Sequencing-Based Method.

Mycobacteriophages are viruses that specifically infect mycobacteria and which, due to their diversity, represent a large gene pool. Characterization of the function of these genes should provide useful insights into host-phage interactions. Here, we describe a next-generation sequencing (NGS)-based, high-throughput screening approach for the identification of mycobacteriophage-encoded proteins that are toxic to mycobacteria. A plasmid-derived library representing the mycobacteriophage TM4 genome was constructed and transformed into Mycobacterium smegmatis. NGS and growth assays showed that the expression of TM4 gp43, gp77, -78, and -79, or gp85 was toxic to M. smegmatis. Although the genes associated with bacterial toxicity were expressed during phage infection, they were not required for lytic replication of mycobacteriophage TM4. In conclusion, we describe here an NGS-based approach which required significantly less time and resources than traditional methods and allowed the identification of novel mycobacteriophage gene products that are toxic to mycobacteria. IMPORTANCE The wide spread of drug-resistant Mycobacterium tuberculosis has brought an urgent need for new drug development. Mycobacteriophages are natural killers of M. tuberculosis, and their toxic gene products might provide potential anti-M. tuberculosis candidates. However, the enormous genetic diversity of mycobacteriophages poses challenges for the identification of these genes. Here, we used a simple and convenient screening method, based on next-generation sequencing, to identify mycobacteriophage genes encoding toxic products for mycobacteria. Using this approach, we screened and validated several toxic products encoded by mycobacteriophage TM4. In addition, we also found that the genes encoding these toxic products are nonessential for lytic replication of TM4. Our work describes a promising method for the identification of phage genes that encode proteins that are toxic to mycobacteria and which might facilitate the identification of novel antimicrobial molecules.

Exploring Host-Binding Machineries of Mycobacteriophages with AlphaFold2.

Although more than 12,000 bacteriophages infecting mycobacteria (mycobacteriophages) have been isolated so far, there is a knowledge gap on their structure-function relationships. Here, we have explored the architecture of host-binding machineries from seven representative mycobacteriophages of the Siphoviridae family infecting Mycobacterium smegmatis, Mycobacterium abscessus, and Mycobacterium tuberculosis, using AlphaFold2 (AF2). AF2 enables confident structural analyses of large and flexible biological assemblies resistant to experimental methods, thereby opening new avenues to shed light on phage structure and function. Our results highlight the modularity and structural diversity of siphophage host-binding machineries that recognize host-specific receptors at the onset of viral infection. Interestingly, the studied mycobacteriophages' host-binding machineries present unique features compared with those of phages infecting other Gram-positive actinobacteria. Although they all assemble the classical Dit (distal tail), Tal (tail-associated lysin), and receptor-binding proteins, five of them contain two potential additional adhesion proteins. Moreover, we have identified brush-like domains formed of multiple polyglycine helices which expose hydrophobic residues as potential receptor-binding domains. These polyglycine-rich domains, which have been observed in only five native proteins, may be a hallmark of mycobacteriophages' host-binding machineries, and they may be more common in nature than expected. Altogether, the unique composition of mycobacteriophages' host-binding machineries indicate they might have evolved to bind to the peculiar mycobacterial cell envelope, which is rich in polysaccharides and mycolic acids. This work provides a rational framework to efficiently produce recombinant proteins or protein domains and test their host-binding function and, hence, to shed light on molecular mechanisms used by mycobacteriophages to infect their host. IMPORTANCE Mycobacteria include both saprophytes, such as the model system Mycobacterium smegmatis, and pathogens, such as Mycobacterium tuberculosis and Mycobacterium abscessus, that are poorly responsive to antibiotic treatments and pose a global public health problem. Mycobacteriophages have been collected at a very large scale over the last decade, and they have proven to be valuable tools for mycobacteria genetic manipulation, rapid diagnostics, and infection treatment. Yet, molecular mechanisms used by mycobacteriophages to infect their host remain poorly understood. Therefore, exploring the structural diversity of mycobacteriophages' host-binding machineries is important not only to better understand viral diversity and bacteriophage-host interactions, but also to rationally develop biotechnological tools. With the powerful protein structure prediction software AlphaFold2, which was publicly released a year ago, it is now possible to gain structural and functional insights on such challenging assemblies.

A novel nucleic acid-binding protein, Gp49, from mycobacteriophage with mycobactericidal activity has the potential to be a therapeutic agent.

The mycobacteriophages encode unique proteins that are potent to be therapeutic agents. We screened several clones with mycobactericidal properties from a genomic library of mycobacteriophages. Here we report the properties of one such clone coding the gene product, Gp49, of the phage Che12. Gp49 is a 16 kD dimeric protein having an HTH motif at its C-terminal and is highly conserved among mycobacteriophages and likely to be part of phage DNA replication machinery. Alphafold predicts it to be an α-helical protein. However, its CD spectrum showed it to be predominantly ß-sheeted. It is a high-affinity heparin-binding protein having similarities with the macrophage protein Azurocidin. Its ß-sheeted apo-structure gets transformed into α-helix upon binding to heparin. It binds to linear dsDNA as well as ssDNA and RNA cooperatively in a sequence non-specific manner. This DNA binding property enables it to inhibit both in vitro and in vivo transcription. The c-terminal HTH motif is responsible for binding to both heparin and nucleic acids. Its in vivo localization on DNA could cause displacements of many DNA-binding proteins from the bacterial chromosome. We surmised that the bactericidal activity of Gp49 arises from its non-specific DNA binding leading to the inhibition of many host-DNA-dependent processes. Its heparin-binding ability could have therapeutic/diagnostic usages in bacterial sepsis treatment.

Characterization and genome analysis of G1 sub-cluster mycobacteriophage Lang.

Phage therapy is revitalized as an alternative to antibiotics therapy against antimicrobials resistant pathogens. Mycobacteriophages are genetically diverse viruses that can specifically infect Mycobacterium genus including Mycobacterium tuberculosis and Mycobacterium smegmatis. Here, we isolated and annotated the genome of a mycobacteriophage Lang, a temperate mycobacteriophage isolated from the soil of Hohhot, Inner Mongolia, China, by using Mycolicibacterium smegmatis mc2 155 as the host. It belongs to the Siphoviridae family of Caudovirales as determined by transmission electron microscopy. The morphological characteristics and certain biological properties of the phage were considered in detail. Phage Lang genomes is 41,487 bp in length with 66.85% GC content and encodes 60 putative open reading frames and belongs to the G1 sub-cluster. Genome annotation indicated that genes for structure proteins, assembly proteins, replications/transcription and lysis of the host are present in function clucters. The genome sequence of phage Lang is more than 95% similar to that of mycobacteriophage Grizzly and Sweets, differing in substitutions, insertions and deletions in Lang. One-step growth curve revealed that Lang has a latent period of 30 min and a outbreak period of 90 min. The short latent period and rapid outbreak mark the unique properties of phage Lang, which can be another potential source for combating M. tuberculosis.

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.

Phylogenomic analyses and host range prediction of cluster P mycobacteriophages.

Bacteriophages, infecting bacterial hosts in every environment on our planet, are a driver of adaptive evolution in bacterial communities. At the same time, the host range of many bacteriophages-and thus one of the selective pressures acting on complex microbial systems in nature-remains poorly characterized. Here, we computationally inferred the putative host ranges of 40 cluster P mycobacteriophages, including members from 6 subclusters (P1-P6). A series of comparative genomic analyses revealed that mycobacteriophages of subcluster P1 are restricted to the Mycobacterium genus, whereas mycobacteriophages of subclusters P2-P6 are likely also able to infect other genera, several of which are commonly associated with human disease. Further genomic analysis highlighted that the majority of cluster P mycobacteriophages harbor a conserved integration-dependent immunity system, hypothesized to be the ancestral state of a genetic switch that controls the shift between lytic and lysogenic life cycles-a temperate characteristic that impedes their usage in antibacterial applications.

Mycobacteriophages: From Petri dish to patient.

Mycobacteriophages-bacteriophages infecting Mycobacterium hosts-contribute substantially to our understanding of viral diversity and evolution, provide resources for advancing Mycobacterium genetics, are the basis of high-impact science education programs, and show considerable therapeutic potential. Over 10,000 individual mycobacteriophages have been isolated by high school and undergraduate students using the model organism Mycobacterium smegmatis mc2155 and 2,100 have been completely sequenced, giving a high-resolution view of the phages that infect a single common host strain. The phage genomes are revealed to be highly diverse and architecturally mosaic and are replete with genes of unknown function. Mycobacteriophages have provided many widely used tools for Mycobacterium genetics including integration-proficient vectors and recombineering systems, as well as systems for efficient delivery of reporter genes, transposons, and allelic exchange substrates. The genomic insights and engineering tools have facilitated exploration of phages for treatment of Mycobacterium infections, although their full therapeutic potential has yet to be realized.

A monomeric mycobacteriophage immunity repressor utilizes two domains to recognize an asymmetric DNA sequence.

Regulation of bacteriophage gene expression involves repressor proteins that bind and downregulate early lytic promoters. A large group of mycobacteriophages code for repressors that are unusual in also terminating transcription elongation at numerous binding sites (stoperators) distributed across the phage genome. Here we provide the X-ray crystal structure of a mycobacteriophage immunity repressor bound to DNA, which reveals the binding of a monomer to an asymmetric DNA sequence using two independent DNA binding domains. The structure is supported by small-angle X-ray scattering, DNA binding, molecular dynamics, and in vivo immunity assays. We propose a model for how dual DNA binding domains facilitate regulation of both transcription initiation and elongation, while enabling evolution of other superinfection immune specificities.

Systematic overexpression of genes encoded by mycobacteriophage Waterfoul reveals novel inhibitors of mycobacterial growth.

Bacteriophages represent an enormous reservoir of novel genes, many of which are unrelated to existing entries in public databases and cannot be assigned a predicted function. Characterization of these genes can provide important insights into the intricacies of phage-host interactions and may offer new strategies to manipulate bacterial growth and behavior. Overexpression is a useful tool in the study of gene-mediated effects, and we describe here the construction of a plasmid-based overexpression library of a complete set of genes for Waterfoul, a mycobacteriophage closely related to those infecting clinically important strains of Mycobacterium tuberculosis and/or Mycobacterium abscessus. The arrayed Waterfoul gene library was systematically screened in a plate-based cytotoxicity assay, identifying a diverse set of 32 Waterfoul gene products capable of inhibiting the growth of the host Mycobacterium smegmatis and providing a first look at the frequency and distribution of cytotoxic products encoded within a single mycobacteriophage genome. Several of these Waterfoul gene products were observed to confer potent anti-mycobacterial effects, making them interesting candidates for follow-up mechanistic studies.

Apoptosis like symptoms associated with abortive infection of Mycobacterium smegmatis by mycobacteriophage D29.

Mycobacteriophages are phages that infect mycobacteria resulting in their killing. Although lysis is the primary mechanism by which mycobacteriophages cause cell death, others such as abortive infection may also be involved. We took recourse to perform immunofluorescence and electron microscopic studies using mycobacteriophage D29 infected Mycobacterium smegmatis cells to investigate this issue. We could observe the intricate details of the infection process using these techniques such as adsorption, the phage tail penetrating the thick mycolic acid layer, formation of membrane pores, membrane blebbing, and phage release. We observed a significant increase in DNA fragmentation and membrane depolarization using cell-biological techniques symptomatic of programmed cell death (PCD). As Toxin-Antitoxin (TA) systems mediate bacterial PCD, we measured their expression profiles with and without phage infection. Of the three TAs examined, MazEF, VapBC, and phd/doc, we found that in the case of VapBC, a significant decrease in the antitoxin (VapB): toxin (VapC) ratio was observed following phage infection, implying that high VapC may have a role to play in the induction of mycobacterial apoptotic cell death following phage infection. This study indicates that D29 infection causes mycobacteria to undergo morphological and molecular changes that are hallmarks of apoptotic cell death.

Use of uncoated magnetic beads to capture Mycobacterium smegmatis and Mycobacterium avium paratuberculosis prior detection by mycobacteriophage D29 and real-time-PCR.

Uncoated tosyl-activated magnetic beads were evaluated to capture Mycobacterium smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) from spiked feces, milk, and urine. Centrifugation and uncoated magnetic beads recovered more than 99% and 93%, respectively, of 1.68 × 107 CFU/mL, 1.68 × 106 CFU/mL and 1.68 × 105 CFU/mL M. smegmatis cells resuspended in phosphate buffer saline. The use of magnetic beads was more efficient to concentrate cells from 1.68 × 104 CFU/mL of M. smegmatis than centrifugation. Likewise, the F57-qPCR detection of MAP cells was different whether they were recovered by beads or centrifugation; cycle threshold (Ct) was lower (p < 0.05) for the detection of MAP cells recovered by beads than centrifugation, indicative of greater recovery. Magnetic separation of MAP cells from milk, urine, and feces specimens was demonstrated by detection of F57 and IS900 sequences. Beads captured no less than 109 CFU/mL from feces and no less than 104 CFU/mL from milk and urine suspensions. In another detection strategy, M. smegmatis coupled to magnetic beads were infected by mycobacteriophage D29. Plaque forming units were observed after 24 h of incubation from urine samples containing 2 × 105 and 2 × 103 CFU/mL M. smegmatis. The results of this study provide a promising tool for diagnosis of tuberculosis and Johne's disease.

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.