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.

Whole genome sequencing identifies an allele responsible for clear vs. turbid plaque morphology in a Mycobacteriophage.

BACKGROUND: Whole genome sequencing promises to revolutionize our ability to link genotypic and phenotypic variation in a wide range of model and non-model species. RESULTS: Here we describe the isolation and characterization of a novel mycobacteriophage named BGlluviae that grows on Mycobacterium smegmatis mc2155. BGlluviae normally produces turbid plaques but a spontaneous clear plaque was also recovered. The genomic DNA from pure populations of the BGlluviae phage and the clear plaque mutant were sequenced. A single substitution, at amino acid 54 (I to T), in the immunity repressor protein resulted in a clear plaque phenotype. CONCLUSIONS: This substitution is predicted to be located at the subunit interaction interface of the repressor protein, and thus prevents the establishment of lysogeny.

A mycobacteriophage genomics approach to identify novel mycobacteriophage proteins with mycobactericidal properties.

Mycobacteriophages that are specific to mycobacteria are sources of various effector proteins that are capable of eliciting bactericidal responses. We describe a genomics approach in combination with bioinformatics to identify mycobacteriophage proteins that are toxic to mycobacteria upon expression. A genomic library comprising phage genome collections was screened for clones capable of killing Mycobacterium smegmatis strain mc2155. We identified four unique clones: clones 45 and 12N (from the mycobacteriophage D29) and clones 66 and 85 (from the mycobacteriophage Che12). The gene products from clones 66 and 45 were identified as Gp49 of the Che12 phage and Gp34 of the D29 phage, respectively. The gene products of the other two clones, 85 and 12N, utilized novel open reading frames (ORFs) coding for synthetic proteins. These four clones (clones 45, 66, 85 and 12N) caused growth defects in M. smegmatis and Mycobacterium bovis upon expression. Clones with Gp49 and Gp34 also induced growth defects in Escherichia coli, indicating that they target conserved host machineries. Their expression induced various morphological changes, indicating that they affected DNA replication and cell division steps. We predicted that Gp34 is a Xis protein that is required in phage DNA excision from the bacterial chromosome. Gp49 is predicted to have an HTH motif with DNA-bending/twisting properties. We suggest that this methodology is useful to identify new phage proteins with the desired properties without laboriously characterizing the individual phages. It is universal and could be applied to other bacteria-phage systems. We speculate that the existence of a virtually unlimited number of phages with unique gene products could offer a cheaper and less hazardous alternative to explore new antimicrobial molecules.

Recovery of mycobacteriophages from archival stocks stored for approximately 50 years in Japan.

Mycobacteriophage archival stocks have been kept for ca. 20-50 years in Japan. In this study, we attempted to recover mycobacteriophages from 50 archival stocks and briefly analyzed the recovered phages. The phages were recovered from 72.2% (13/18) of the lyophilized stocks that had been stored for 47-56 years. Moreover, the analysis of 12 representative recovered phages led to their classification as belonging to the family Siphoviridae, and seven of them were typed by polymerase chain reaction (PCR) targeting the gene that encodes the tape measure protein. Considering these results, lyophilization seems to be suitable for phage archival storage.

Isolation and characterization of bacteriophages from India, with lytic activity against Mycobacterium tuberculosis.

Bacteriophages are being considered as a promising natural resource for the development of alternative strategies against mycobacterial diseases, especially in the context of the wide-spread occurrence of drug resistance among the clinical isolates of Mycobacterium tuberculosis. However, there is not much information documented on mycobacteriophages from India. Here, we report the isolation of 17 mycobacteriophages using Mycobacterium smegmatis as the bacterial host, where 9 phages also lyse M. tuberculosis H37Rv. We present detailed analysis of one of these mycobacteriophages - PDRPv. Transmission electron microscopy and polymerase chain reaction analysis (of a conserved region within the TMP gene) show PDRPv to belong to the Siphoviridae family and B1 subcluster, respectively. The genome (69 110 bp) of PDRPv is circularly permuted double-stranded DNA with ∼66% GC content and has 106 open reading frames (ORFs). On the basis of sequence similarity and conserved domains, we have assigned function to 28 ORFs and have broadly categorized them into 6 groups that are related to replication and genome maintenance, DNA packaging, virion release, structural proteins, lysogeny-related genes and endolysins. The present study reports the occurrence of novel antimycobacterial phages in India and highlights their potential to contribute to our understanding of these phages and their gene products as potential antimicrobial agents.

Complete genome sequence analysis of the novel mycobacteriophage Shandong1.

In this study, we isolated a mycobacteriophage infecting Mycobacterium smegmatis mc2155 from a soil sample collected in Shandong Province in China. This phage was named Shandong1. It is a member of the family Siphoviridae with an isometric head and a long tail. Its genome was found to be 60,618 bp long with 67.46% G + C content and 96 putative protein-coding genes. No tRNA-encoding genes were identified. Comparative genomics analysis showed that the mycobacteriophage Shandong1 should be considered a member of a new species in mycobacteriophage cluster K.

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.

Genome sequence of a cluster A13 mycobacteriophage detected in Mycobacterium phlei over a half century ago.

A phage infecting Mycobacterium phlei was isolated in 1958 from a soil sample in Hungary. Some physicochemical and biological properties of the virus were described in independent studies over the years. Here, we report the genome sequence of this early mycobacteriophage isolate. The Phlei phage genome measured 50,418 bp, had a GC content of 60.1 % and was predicted to encode 81 proteins and three tRNAs. Phylogeny of the tape measure protein revealed genetic relatedness to other early isolates of mycobacteriophages within subcluster A2. The genomic organization and genetic relationships to other strains showed that the Phlei phage belongs to a novel genetic cluster, designated A13.

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.

Biology of a novel mycobacteriophage, SWU1, isolated from Chinese soil as revealed by genomic characteristics.

Mycobacteriophage SWU1 is a newly isolated phage from a soil sample collected at Gongping village, Pingchang County, Sichuan Province, China, using Mycobacterium smegmatis mc(2)155 as a host. Plaques of SWU1 appear as a unique bull's-eye on an M. smegmatis lawn. In this paper, we report the complete genome sequence of SWU1 and some major findings from the analysis result.

Complete genome sequences of 138 mycobacteriophages.

Bacteriophages are the most numerous biological entities in the biosphere, and although their genetic diversity is high, it remains ill defined. Mycobacteriophages-the viruses of mycobacterial hosts-provide insights into this diversity as well as tools for manipulating Mycobacterium tuberculosis. We report here the complete genome sequences of 138 new mycobacteriophages, which-together with the 83 mycobacteriophages previously reported-represent the largest collection of phages known to infect a single common host, Mycobacterium smegmatis mc(2) 155.

Mycobacteriophage Marvin: a new singleton phage with an unusual genome organization.

Mycobacteriophages represent a genetically diverse group of viruses that infect mycobacterial hosts. Although more than 80 genomes have been sequenced, these still poorly represent the likely diversity of the broader population of phages that can infect the host, Mycobacterium smegmatis mc(2)155. We describe here a newly discovered phage, Marvin, which is a singleton phage, having no previously identified close relatives. The 65,100-bp genome contains 107 predicted protein-coding genes arranged in a noncanonical genomic architecture in which a subset of the minor tail protein genes are displaced about 20 kbp from their typical location, situated among nonstructural genes anticipated to be expressed early in lytic growth. Marvin is not temperate, and stable lysogens cannot be recovered from infections, although the presence of a putative xis gene suggests that Marvin could be a relatively recent derivative of a temperate parent. The Marvin genome is replete with novel genes not present in other mycobacteriophage genomes, and although most are of unknown function, the presence of amidoligase and glutamine amidotransferase genes suggests intriguing possibilities for the interactions of Marvin with its mycobacterial hosts.

Impact of relative humidity and collection media on mycobacteriophage D29 aerosol.

This study was conducted to evaluate the effect of aerosol generation, methods of sampling, storage conditions, and relative humidity on the culturability of the mycobacteriophage D29. The lytic phage D29 can kill Mycobacterium tuberculosis, and the phage aerosol can be treated as a potential tool for tuberculosis treatment. The culturability of D29 was tested using a test chamber designed for the bioaerosols research against three spray liquids (deionized water, phosphate-buffered saline [PBS], and normal saline), four collection media (suspension medium [SM], nutrient broth, PBS, and deionized water), two sampling systems (the all-glass impinger AGI-30 and the Biosampler) and across a range of humidities (20 to 90%). The effect of storage conditions on the culturability of collected sample was also evaluated for the AGI-30 impinger. The results proved that viable phage D29 particles generated by deionized water were approximately 30- and 300-fold higher than PBS and normal saline, respectively. As collection media, SM buffer and nutrient broth were observed to yield a higher number of plaques compared to PBS and deionized water. No difference was observed in collection efficiency between AGI-30 and Biosampler with two detection methods (culture-based technique and real-time PCR). The culturability of collected D29 in SM buffer or nutrient broth can be maintained up to 12 h irrespective of storage temperature. Relative humidity was found to strongly influence airborne D29 culturability which is 2- to 20-fold higher in low humidity (25%) than medium (55%) or high (85%) humidity. This research will help identify the optimal means for the application of D29 aerosol in animal inhalation experiments.

Genomic and proteomic portrait of a novel mycobacteriophage SWU2 isolated from China.

Phage therapy, especially combination with antibiotics, was revitalized to control the antibiotics resistance. Mycobacteriophage, the phage of mycobacterium with the most notorious Mycobacterium tuberculosis (M. tuberculosis), was intensively explored. A novel mycobacteriophage SWU2 was isolated from a soil sample collected at Nanchang city, Jiangxi province, China, by using Mycolicibacterium smegmatis (M. smegmatis) mc2 155 as the host. Phage morphology and biology were characterized. Phage structure proteins were analyzed by LC-MS/MS. The putative functions of phage proteins and multi-genome comparison were performed with bioinformatics. The transmission electron microscopy result indicated that this phage belongs to Siphoviridae of Caudovirales. Plaques of SWU2 appeared clear but small. In a one-step growth test, we demonstrated that SWU2 had a latent period of 30 min and a logarithmic phase of 120 min. Among the 76 predicted Open Reading Frames (ORFs), 9 ORFs were identified as phage structure proteins of SWU2. The assembled phage genome size is 50,013 bp, with 62.7% of G + C content. SWU2 genome sequence shares 88% identity with Mycobacterium phages HINdeR and Timshel, differing in substitutions, insertions and deletions in SWU2. Phylogenetic tree revealed that SWU2 is grouped into A7 sub-cluster. There are several substitutions, insertions and deletions in SWU2 genome in comparison with close cousin phages HINdeR and Timshel. The new phage adds another dimension of abundance to the mycobacteriophages.

Weirdo19ES is a novel singleton mycobacteriophage that selects for glycolipid deficient phage-resistant M. smegmatis mutants.

The sequencing and bioinformatics analysis of bacteriophages infecting mycobacteria has yielded a large amount of information on their evolution, including that on their environmental propagation on other genera such as Gordonia, closely related to Mycobacterium. However, little is known on mycobacteriophages cell biology such as the nature of their receptor(s) or their replication cycle. As part of our on-going screening for novel mycobacteriophages, we herein report the isolation and genome bioinformatics analysis of Weirdo19ES, a singleton Siphoviridae temperate mycobacteriophage with a 70.19% GC content. Nucleotide and protein sequence comparison to actinobacteriophage databases revealed that Weirdo19ES shows low homology to Gordonia phage Ruthy and mycobacteriophages falling in clusters Q and G and to singleton DS6A.Weirdo19ES also displays uncommon features such as a very short Lysin A gene (with only one enzymatic domain) and two putative HNH endonucleases. Mycobacterium smegmatis mutants resistant to Weirdo19ES are cross- resistant to I3. In agreement with that phenotype, analysis of cell envelope of those mutants showed that Weirdo19ES shares receptors with the transducing mycobacteriophage I3.This singleton mycobacteriophage adds up to the uncommonness of local mycobacteriophages previously isolated by our group and helps understanding the nature of mycobacteriophage receptors.

Mycobacteriophages as versatile tools for genetic manipulation of mycobacteria and development of simple methods for diagnosis of mycobacterial diseases.

Tuberculosis, caused by Mycobacterium tuberculosis, is responsible for over two million deaths per year worldwide. Due to its long doubling time (18 h), the microbiological detection of M. tuberculosis by conventional methods takes up to one month, unless the number of bacilli in the biological sample is high enough. Thus, drug resistance assessment requires at least one month for obtaining the primary culture and another month to determine its susceptibility to antimycobacterial drugs. Moreover, for a long time, the lack of genetic tools for mycobacteria has been a barrier for undertaking studies aimed at understanding the mechanisms of drug resistance and drug target identification, being all these topics of utmost importance considering the increase in the number of drug-resistant clones and the few therapeutic options available. Mycobacteriophages are promising as a novel source of genetic elements for mycobacteria manipulation, as well as for the development of versatile, simple, fast and cheap methods for drug resistance assessment of M. tuberculosis clinical isolates. We herein describe the background related to the use of mycobacteriophages, with emphasis placed on their utilization for drug resistance analysis in our country.

Mycobacteriophage CRB2 defines a new subcluster in mycobacteriophage classification.

Mycobacteriophages are viruses -mostly temperates- that infect Mycobacterium smegmatis and sometimes Mycobacterium tuberculosis. Mycobacteriophages are grouped in clusters on the basis of the overall nucleotide sequence homology, being further divided in subclusters as more mycobacteriophage genomes are sequenced and annotated. As part of our on-going screening for novel isolates, we herein report the bioinformatics analysis of CRB2, a mycobacteriophage belonging into the Siphoviridae family that propagates at 30°C. CRB2 has a 72,217 bp genome with a 69.78% GC content that belongs to Cluster B; nucleotide comparison with other B cluster members positions CRB2 as the sole member of a new subcluster, B9, being mycobacteriophage Saguaro (belonging into subcluster B7) its closest relative. Sequencing and annotation of 14 mycobacteriophages isolated by our group has yielded six cluster A members, a singleton, four of the five members of subcluster B6, one of the three reported members of subcluster G4, and CRB2 which defines subcluster B9. Considering the massive mycobacteriophage search performed in USA and the relatively rarity of our phages, we propose that factors other than size of the sampling determine the variability of mycobacteriophage distribution, and thus a world-wide concerted mining would most likely bring extremely rare and yet undiscovered mycobacteriophages.

Host range of 14 mycobacteriophages in Mycobacterium ulcerans and seven other mycobacteria including Mycobacterium tuberculosis--application for identification and susceptibility testing.

The host range of well-characterized mycobacteriophages, such as D29 and TM4, has been determined, together with that of more recently isolated mycobacteriophages, in Mycobacterium ulcerans, Mycobacterium tuberculosis, Mycobacterium bovis BCG, Mycobacterium avium, Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacterium fortuitum and Mycobacterium chelonae. Here, a set of virulent phages for M. ulcerans, a pathogen with a dramatic increase of incidence over the last decade, is demonstrated. In this work, a mycobacteriophage replication assay was adapted for the identification and rifampicin-susceptibility testing of M. ulcerans. Mycobacteriophages have generated a number of useful tools and enabled insights into mycobacterial genetics. With regard to the neglected pathogen M. ulcerans, the findings presented in this work allow the application of a large range of phage-based vectors and markers. The potential of phage therapy can now be evaluated for this extracellular pathogen.

Comparison of redox and D29 phage methods for detection of isoniazid and rifampicin resistance in Mycobacterium tuberculosis.

Rapid, accurate and inexpensive methods are essential to detect drug-resistant Mycobacterium tuberculosis and allow timely application of effective treatment and precautions to prevent transmission. The proportion method, the MTT and Alamar Blue redox methods, and the D29 mycobacteriophage assay, were compared for their ability to detect resistance to isoniazid and rifampicin. When tested against a panel of known M. tuberculosis strains, the redox methods and the D29 assay showed good sensitivity and specificity compared to the proportion method, suggesting that they could be useful alternatives for identifying multidrug resistance in M. tuberculosis.

Different characteristics between mycobacteriophage Chy1 and D29, which were classified as cluster A2 mycobacteriophages.

PURPOSE: The aim of this study was to isolate a novel mycobacteriophage and then explore its anti-tuberculosis (TB) potential. MATERIALS AND METHODS: Phage was isolated from enriched soil sample. A total of 36 mycobacterial strains obtained from clinical specimens were subjected to investigate the host range of phage by the spot lysis assay. Biological characteristics were investigated through growth curve, host range and phage antimicrobial activity in vitro. Then, genome sequencing and further analysis were accomplished by using an ABI3730XL DNA sequencer and comparative genome, respectively. RESULTS: A lytic mycobacteriophage (Chy1) was isolated and the plaque morphology was similar to D29. The genome of Chy1 was estimated to be about 47,198 base pair (bp) and strong similarity (97.4% identity) to D29, especially, the Chy1 gene 7 encoding holin which is considered as a clock controlling growth cycle of the corresponding phage, was identical (100% identity) to phage D29 gene 11, thus classifying Chy1 as a member of the cluster A2 family. However, to our surprise, Chy1 can infect a narrower range of host-mycobacterial strains than that of D29. The latent period of Chy1 was quite longer compared to D29. Moreover, Chy1 has a weaker ability to lyse Mycobacterium smegmatis compared to D29. CONCLUSIONS: The sequence of Chy1 showed 97.4% homology with the genome sequence of D29, but there was a large difference in their biological characteristics. Overall, the results of this investigation indicate that Chy1 is not an ideal candidate for developing mycobacteriophage-based anti-TB therapies but for future researches to investigate the reason why biological characteristics of Chy1 and D29 were remarkably different.