Mycobacteriophage-repressor-mediated immunity as a selectable genetic marker: Adephagia and BPs repressor selection.

Mycobacteriophages provide an abundance of systems for use in mycobacterial genetics, including manipulation of Mycobacterium tuberculosis. Because of the dearth of antibiotic resistance cassettes and biosafety concerns in constructing recombinant virulent M. tuberculosis strains, we developed the use of mycobacteriophage-encoded repressor genes that can be selected in the presence of lytic versions of their cognate phages. The phage Adephagia repressor gene (43) was identified through its ability to confer immunity to Adephagia superinfection, together with the mapping of mutations in gene 43 that confer a clear-phage phenotype. Plasmid transformants containing either Adephagia 43 or the previously identified BPs repressor 33 can be readily selected following electroporation using engineered lytic derivatives of Adephagia and BPs, respectively. Selection is as efficient as antibiotic selection, can be used with either single-copy integration vectors or with extrachromosomal vectors, and works similarly in both Mycobacterium smegmatis and M. tuberculosis.

Development of a novel phage-mediated immunoassay for the rapid detection of viable Mycobacterium avium subsp. paratuberculosis.

AIMS: The objective of this study was to develop a novel screening method for detection of viable Mycobacterium avium subsp. paratuberculosis (Map) in milk and faeces, as a rapid alternative to Map culture. METHODS AND RESULTS: The new method couples Map-specific peptide-mediated magnetic separation technique with an optimized phage amplification assay followed by detection of released progeny phage by ELISA in a competition assay format using polyclonal antibody produced against the D29 mycobacteriophage involved in the phage assay. Sample matrices were found not to interfere with the developed method, and the dynamic range of the assay was 3 × 10(2) -6 × 10(8 ) phage ml(-1) . When low numbers of Map were present (10(2)  CFU ml(-1) ), the burst size of a single host Map cell was maximal (10(3) phage per cell) resulting in a highly sensitive screening assay. CONCLUSION: A rapid, sensitive immuno-based screening method suitable for the detection of viable Map in milk and faeces was developed. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel PMS-phage-ELISA permits sensitive, qualitative detection of viable Map in milk or faeces samples within 48 h, representing a substantial decrease in time to detection compared with current culture methods for Map.

Evolution of Superinfection Immunity in Cluster A Mycobacteriophages.

Temperate phages encode an immunity system to control lytic gene expression during lysogeny. This gene regulatory circuit consists of multiple interacting genetic elements, and although it is essential for controlling phage growth, it is subject to conflicting evolutionary pressures. During superinfection of a lysogen, the prophage's circuit interacts with the superinfecting phage's circuit and prevents lytic growth if the two circuits are closely related. The circuitry is advantageous since it provides the prophage with a defense mechanism, but the circuitry is also disadvantageous since it limits the phage's host range during superinfection. Evolutionarily related phages have divergent, orthogonal immunity systems that no longer interact and are heteroimmune, but we do not understand how immunity systems evolve new specificities. Here, we use a group of Cluster A mycobacteriophages that exhibit a spectrum of genetic diversity to examine how immunity system evolution impacts superinfection immunity. We show that phages with mesotypic (i.e., genetically related but distinct) immunity systems exhibit asymmetric and incomplete superinfection phenotypes. They form complex immunity networks instead of well-defined immunity groups, and mutations conferring escape (i.e., virulence) from homotypic or mesotypic immunity have various escape specificities. Thus, virulence and the evolution of new immune specificities are shaped by interactions with homotypic and mesotypic immunity systems.IMPORTANCE Many aspects regarding superinfection, immunity, virulence, and the evolution of immune specificities are poorly understood due to the lack of large collections of isolated and sequenced phages with a spectrum of genetic diversity. Using a genetically diverse collection of Cluster A phages, we show that the classical and relatively straightforward patterns of homoimmunity, heteroimmunity, and virulence result from interactions between homotypic and heterotypic phages at the extreme edges of an evolutionary continuum of immune specificities. Genetic interactions between mesotypic phages result in more complex mesoimmunity phenotypes and virulence profiles. These results highlight that the evolution of immune specificities can be shaped by homotypic and mesotypic interactions and may be more dynamic than previously considered.

Mycobacteriophage ZoeJ: A broad host-range close relative of mycobacteriophage TM4.

A collection of over 1600 sequenced bacteriophages isolated on a single host strain, Mycobacterium smegmatis mc2155, can be grouped into over two dozen types that have little or no nucleotide sequence similarity to each other. One group, Cluster K, can be divided into several subclusters, and the well-characterized and much exploited phage TM4 lies in Subcluster K2. Many of the Cluster K phages have broad host ranges and infect both fast- and slow-growing mycobacterial strains. Here we describe phage ZoeJ, a new Subcluster K2 member, which infects a broad spectrum of mycobacterial hosts including M. smegmatis, Mycobacterium tuberculosis, and Mycobacterium avium. ZoeJ has extensive sequence similarity to TM4, and comparative analysis reveals the precise deletion conferring the lytic phenotype of TM4. The ZoeJ immunity repressor was identified as gene 45, which is prophage-expressed, is required for lysogeny, and is sufficient to confer superinfection immunity to ZoeJ. ZoeJ gp45 also confers immunity to Subcluster K2 phage Milly, and Subcluster K1 phages Adephagia and CrimD, but surprisingly not to TM4. RNAseq analysis reveals the temporal pattern of early and late gene expressions in ZoeJ lytic growth and suggests a role for the ESAS motifs for gene regulation.

An Unusual Phage Repressor Encoded by Mycobacteriophage BPs.

Temperate bacteriophages express transcription repressors that maintain lysogeny by down-regulating lytic promoters and confer superinfection immunity. Repressor regulation is critical to the outcome of infection-lysogenic or lytic growth-as well as prophage induction into lytic replication. Mycobacteriophage BPs and its relatives use an unusual integration-dependent immunity system in which the phage attachment site (attP) is located within the repressor gene (33) such that site-specific integration leads to synthesis of a prophage-encoded product (gp33103) that is 33 residues shorter at its C-terminus than the virally-encoded protein (gp33136). However, the shorter form of the repressor (gp33103) is stable and active in repression of the early lytic promoter PR, whereas the longer virally-encoded form (gp33136) is inactive due to targeted degradation via a C-terminal ssrA-like tag. We show here that both forms of the repressor bind similarly to the 33-34 intergenic regulatory region, and that BPs gp33103 is a tetramer in solution. The BPs gp33103 repressor binds to five regulatory regions spanning the BPs genome, and regulates four promoters including the early lytic promoter, PR. BPs gp33103 has a complex pattern of DNA recognition in which a full operator binding site contains two half sites separated by a variable spacer, and BPs gp33103 induces a DNA bend at the full operator site but not a half site. The operator site structure is unusual in that one half site corresponds to a 12 bp palindrome identified previously, but the other half site is a highly variable variant of the palindrome.

Selective identification of new therapeutic targets of Mycobacterium tuberculosis by IVIAT approach.

The in vivo induced antigen technology (IVIAT)(1) has been used for the identification of open reading frames (ORFs) which could be possible therapeutic targets. A recombinant lambdagt11:: Mycobacterium tuberculosis H37Rv expression library was screened with pooled TB patient sera preabsorbed with in vitro grown M. tuberculosis H37Rv. Preabsorption of pooled TB patient sera allowed identification of antigens specifically expressed or upregulated during infection and growth in vivo. Six ORFs were identified, of which four (rv0287, rv2402, rv3878 and rv1045) were of hypothetical functions. Rv0287 is a probable regulatory protein. Rv3878 is present uniquely in M. tuberculosis H37Rv and is a part of RDI deletion region of M. bovis BCG, which includes esat 6 region. This could be exploited as a tool for diagnosis. Two ORFs were assigned function solely on the basis of homology, dnaQ (rv3711c) and lpdA (rv3303c). dnaQ codes for the epsilon subunit of DNA polymerase III, which is responsible for the proofreading activity of the complex. lpdA codes for dihydrolipoamide dehydrogenase, which is a part of many multienzyme complexes such as pyruvate dehydrogenase, keto-acid dehydrogenase and alpha-ketoglutarate dehydrogenase. These two enzymes appear to be potential targets for drug development.

Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution.

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.

Transcriptional regulation and immunity in mycobacteriophage Bxb1.

Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis that shares a similar genome organization to mycobacteriophage L5, although the two phages are heteroimmune. We have investigated the regulatory circuitry of Bxb1 and found that it encodes a repressor, gp69, which regulates at least two promoters, an early lytic promoter, Pleft, and the divergent promoter, Pright. Bxb1 gp69 is 41% identical to the L5 repressor (gp71) and binds to repressor binding sites that conform to a similar, but distinct, 13 bp asymmetric consensus sequence to that for the L5 gp71 binding sites. The two phage repressors have a strong preference for their cognate binding sites, thus accounting for their immunity phenotypes. The Bxb1 genome contains 34 putative repressor binding sites located throughout the genome, but situated within short intergenic spaces and orientated in only one direction relative to the direction of transcription. Comparison with the locations of repressor binding sites within the L5 genome provides insights into how these unusual regulatory systems evolve.