Complete genome of a novel mycobacteriophage WXIN isolated in Wuhan, China.

OBJECTIVES: The rising of antibiotic resistance has sparked a renewed interest in mycobacteriophage as alternative therapeutic strategies against mycobacterial infections. So far, the vast majority of mycobacteriophages have been isolated using the model species Mycobacterium smegmatis, implying an overwhelming majority of mycobacteriophages in the environment remain uncultured, unclassified, and their specific hosts and infection strategies are still unknown. This study was undertaken to isolate and characterize novel mycobacteriophages targeting Mycobacterium septicum. DATA DESCRIPTION: Here a novel mycobacteriophage WXIN against M. septicum was isolated from soil samples in Wuhan, China. Whole genome analysis indicates that the phage genome consists of 115,158 bp with a GC content of 61.9%. Of the 260 putative open reading frames, 46 may be associated with phage packaging, structure, lysis, lysogeny, genome modification/replication, and other functional roles. The limited genome-wide similarity, along with phylogenetic trees constructed based on viral proteome and orthologous genes show that phage WXIN represents a novel cluster distantly related to cluster J mycobacteriophages (genus Omegavirus). Overall, these results provide novel insights into the genomic properties of mycobacteriophages, highlighting the great genetic diversity of mycobacteriophages in relation to their hosts.

Insights into the genomic features and lifestyle of B1 subcluster mycobacteriophages.

Bacteriophages infecting Mycobacterium smegmatis mc2155 are numerous and, hence, are classified into clusters based on nucleotide sequence similarity. Analyzing phages belonging to clusters/subclusters can help gain deeper insights into their biological features and potential therapeutic applications. In this study, for genomic characterization of B1 subcluster mycobacteriophages, a framework of online tools was developed, which enabled functional annotation of about 55% of the previously deemed hypothetical proteins in B1 phages. We also studied the phenotype, lysogeny status, and antimycobacterial activity of 10 B1 phages against biofilm and an antibiotic-resistant M. smegmatis strain (4XR1). All 10 phages belonged to the Siphoviridae family, appeared temperate based on their spontaneous release from the putative lysogens and showed antibiofilm activity. The highest inhibitory and disruptive effects on biofilm were 64% and 46%, respectively. This systematic characterization using a combination of genomic and experimental tools is a promising approach to furthering our understanding of viral dark matter.

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.

An intramolecular cross-talk in D29 mycobacteriophage endolysin governs the lytic cycle and phage-host population dynamics.

D29 mycobacteriophage encodes LysA endolysin, which mediates mycobacterial host cell lysis by targeting its peptidoglycan layer, thus projecting itself as a potential therapeutic. However, the regulatory mechanism of LysA during the phage lytic cycle remains ill defined. Here, we show that during D29 lytic cycle, structural and functional regulation of LysA not only orchestrates host cell lysis but also is critical for maintaining phage-host population dynamics by governing various phases of lytic cycle. We report that LysA exists in two conformations, of which only one is active, and the protein undergoes a host peptidoglycan-dependent conformational switch to become active for carrying out endogenous host cell lysis. D29 maintains a pool of inactive LysA, allowing complete assembly of phage progeny, thus helping avoid premature host lysis. In addition, we show that the switch reverses after lysis, thus preventing exogenous targeting of bystanders, which otherwise negatively affects phage propagation in the environment.

A novel synergistic enzyme-antibiotic therapy with immobilization of mycobacteriophage Lysin B enzyme onto Rif@UiO-66 nanocomposite for enhanced inhaled anti-TB therapy; Nanoenzybiotics approach.

The emergence of antibiotic-resistant and phage-resistant strains of Mycobacterium tuberculosis (M. tuberculosis) necessitates improving new therapeutic plans. The objective of the current work was to ensure the effectiveness of rifampicin and the mycobacteriophage LysB D29 (LysB)enzyme in the treatment of multi-drug resistant tuberculosis (MDR-TB) infection, where new and safe metal-organic framework (MOF) nanoparticles were used in combination. UiO-66 nanoparticles were synthesized under mild conditions in which the antimycobacterial agent (rifampicin) was loaded (Rif@UiO-66) and LysB D29 enzyme immobilized onto Rif@UiO-66, which were further characterized. Subsequently, the antibacterial activity of different ratios of Rif@UiO-66 and LysB/Rif@uio-66 against the nonpathogenic tuberculosis model Mycobacterium smegmatis (M. smegmatis) was evaluated by minimum inhibitory concentration (MIC) tests. Impressively, the MIC of LysB/Rif@uio-66 was 16-fold lower than that of pure rifampicin. In vitro and in vivo toxicity studies proved that LysB/Rif@UiO-66 is a highly biocompatible therapy for pulmonary infection. A biodistribution assay showed that LysB/Rif@UiO-66 showed a 5.31-fold higher drug concentration in the lungs than free rifampicin. A synergistic interaction between UiO-66, rifampicin and the mycobacteriophage lysB D29 enzyme was shown in the computational method (docking). Therefore, all results indicated that the LysB/Rif@UiO-66 nanocomposite exhibited promising innovative enzyme-antibiotic therapy for tuberculosis treatment.

Alginate-Encapsulated Mycobacteriophage: A Potential Approach for the Management of Intestinal Mycobacterial Disease.

Encapsulated medication is a common method of administering therapeutic treatments. As researchers explore alternative therapies, it is likely that encapsulation will remain a feature of these novel treatments, particularly when routes of delivery are considered. For instance, alginate-encapsulation is often favoured where gastric digestion poses an obstacle. When exposed to cations (namely Ca2+), alginate readily forms gels that are resilient to acidic conditions and readily dissociate in response to mid-range pH. This action can be extremely valuable for the encapsulation of phages. The efficient delivery of phages to the intestine is important when considering mycobacteriophage (MP) therapy (or MP prophylaxis) for disseminated mycobacterial infections and chronic gastroenteritis conditions. This study presents the design and in vitro validation of an alginate-encapsulated MP capable of releasing phages in a pH-dependent manner. Ultimately, it is shown that encapsulated phages pretreated with simulated gastric fluid (SGF) are capable of releasing viable phages into simulated intestinal fluid (SIF) and thereby reducing the mycobacterial numbers in spiked SIF by 90%. These findings suggest that alginate encapsulation may be a viable option for therapeutic and prophylactic approaches to the management of intestinal mycobacterial disease, such as Johne's disease.

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.

Mycobacteriophage D29 Lysin B exhibits promising anti-mycobacterial activity against drug-resistant Mycobacterium tuberculosis.

IMPORTANCE: To combat the rapidly emerging drug-resistant M. tuberculosis, it is now essential to look for alternative therapeutics. Mycobacteriophages can be considered as efficient therapeutics due to their natural ability to infect and kill mycobacteria including M. tuberculosis. Here, we have exploited the mycolyl-arabinogalactan esterase property of LysB encoded from mycobacteriophage D29. This study is novel in terms of targeting a multi-drug-resistant pathogenic strain of M. tuberculosis with LysB and also examining the combination of anti-TB drugs and LysB. All the experiments include external administration of LysB. Therefore, the remarkable lytic activity of LysB overcomes the difficulty to enter the complex cell envelope of mycobacteria. Targeting the intracellularly located M. tuberculosis by LysB and non-toxicity to macrophages take the process of the development of LysB as a drug one step ahead, and also, the interaction studies with rifampicin and isoniazid will help to form a new treatment regimen against tuberculosis.

The Cytotoxic Mycobacteriophage Protein Phaedrus gp82 Interacts with and Modulates the Activity of the Host ATPase, MoxR.

Approximately 70% of bacteriophage-encoded proteins are of unknown function. Elucidating these protein functions represents opportunities to discover new phage-host interactions and mechanisms by which the phages modulate host activities. Here, we describe a pipeline for prioritizing phage-encoded proteins for structural analysis and characterize the gp82 protein encoded by mycobacteriophage Phaedrus. Structural and solution studies of gp82 show it is a trimeric protein containing two domains. Co-precipitation studies with the host Mycobacterium smegmatis identified the ATPase MoxR as an interacting partner protein. Phaedrus gp82-MoxR interaction requires the presence of a loop sequence within gp82 that is highly exposed and disordered in the crystallographic structure. We show that Phaedrus gp82 overexpression in M. smegmatis retards the growth of M. smegmatis on solid medium, resulting in a small colony phenotype. Overexpression of gp82 containing a mutant disordered loop or the overexpression of MoxR both rescue this phenotype. Lastly, we show that recombinant gp82 reduces levels of MoxR-mediated ATPase activity in vitro that is required for its chaperone function, and that the disordered loop plays an important role in this phenotype. We conclude that Phaedrus gp82 binds to and reduces mycobacterial MoxR activity, leading to reduced function of host proteins that require MoxR chaperone activity for their normal activity.

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.

Intrapulmonary Treatment with Mycobacteriophage LysB Rapidly Reduces Mycobacterium abscessus Burden.

Intrinsic and acquired antibiotic resistance in Mycobacterium abscessus presents challenges in infection control, and new therapeutic strategies are needed. Bacteriophage therapy shows promise, but variabilities in M. abscessus phage susceptibility limits its broader utility. We show here that a mycobacteriophage-encoded lysin B (LysB) efficiently and rapidly kills both smooth- and rough-colony morphotype M. abscessus strains and reduces the pulmonary bacterial load in mice. LysB aerosolization presents a plausible treatment for pulmonary M. abscessus 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.