| the cohesive ends of mycobacteriophage l5 dna. | | 1992 | 1620623 |
| development of bcg as a live recombinant vector system: potential use as an hiv vaccine. | bacille calmette-guèrin (bcg), a live attenuated tubercle bacillus, is currently the most widely used vaccine in the world. because of its unique characteristics, including low toxicity, adjuvant potential, and long-lasting immunity, bcg represents a novel vaccine vehicle with which to deliver protective antigens of multiple pathogens. we have developed episomal and integrative expression vectors employing regulatory sequences of major bcg heat shock proteins for stable maintenance and expressio ... | 1991 | 1845119 |
| site-specific integration of mycobacteriophage l5: integration-proficient vectors for mycobacterium smegmatis, mycobacterium tuberculosis, and bacille calmette-guérin. | mycobacteriophage l5, a temperate phage of mycobacteria, integrates site-specifically into the mycobacterium smegmatis chromosome. we have identified the int gene and attp site of l5, characterized the chromosomal attachment site (attb), and constructed plasmid vectors that efficiently transform m. smegmatis through stable site-specific integration of the plasmid into the bacterial genome. these integration-proficient plasmids also efficiently transform slow-growing mycobacteria such as the path ... | 1991 | 1901654 |
| l5 luciferase reporter mycobacteriophages: a sensitive tool for the detection and assay of live mycobacteria. | recombinant bacteriophages provide efficient delivery systems for introducing reporter genes into specific bacterial hosts. we have constructed mycobacteriophage l5 recombinants carrying the firefly luciferase gene inserted into the trna region of the phage genome. infection of mycobacterium smegmatis by these phages results in expression of the luciferase gene and light emission. fortuitously, the luciferase gene is expressed continuously in lysogens surviving infection. synthesis of luciferase ... | 1995 | 7623662 |
| progress on development of the live bcg recombinant vaccine vehicle for combined vaccine delivery. | bcg, the current vaccine for tuberculosis, has been administered to approximately three billion people. this live vaccine has a low incidence of serious side effects and can be given at birth. within the past six years, systems for the manipulation and expression of foreign genes in mycobacteria have been developed, allowing the evaluation of rbcg as a vaccine delivery vehicle for heterologous antigens. recent studies from our group have shown that rbcg expressing outer surface protein a of borr ... | 1995 | 7625654 |
| bacteriophages as tools for vaccine development. | the construction of live recombinant bacterial vaccines requires a reasonably sophisticated genetic system for the introduction, stabilization and expression of foreign antigen genes. bacteriophages offer a rich collection of tools that can be used for vaccine construction, including site-specific integration-proficient vectors, non-antibiotic selectable markers and signals for efficient transcription and translation of foreign genes. we describe the characterization of a temperate phage of the ... | 1994 | 7958482 |
| mycobacteriophage l5 integrase-mediated site-specific integration in vitro. | mycobacteriophage l5, a temperate phage of the mycobacteria, forms stable lysogens in mycobacterium smegmatis via site-specific integration of the phage genome. recombination occurs within specific phage and bacterial attachment sites and is catalyzed by the phage-encoded integrase protein in vivo. we describe here the overexpression and purification of l5 integrase and its ability to mediate integrative recombination in vitro. we find that l5 integrase-mediated recombination is greatly stimulat ... | 1993 | 8226625 |
| dna sequence, structure and gene expression of mycobacteriophage l5: a phage system for mycobacterial genetics. | genetic studies of mycobacterium tuberculosis and other mycobacterial pathogens have suffered from the lack of a sophisticated genetic system. to address this issue we have developed a viral system through a detailed characterization of mycobacteriophage l5, a temperate phage that infects both fast- and slow-growing mycobacteria. we describe here the complete dna sequence of the l5 genome and initial characterization of l5 virion structure and gene expression. in addition to providing a genetic ... | 1993 | 8459766 |
| superinfection immunity of mycobacteriophage l5: applications for genetic transformation of mycobacteria. | mycobacteriophage l5 is a temperate phage of the mycobacteria that forms stable lysogens in mycobacterium smegmatis. we show here that the 183-amino-acid product of l5 gene 71 confers immunity to l5 superinfection, is required for maintenance of the lysogenic state and contains a helix-turn-helix dna-binding motif--properties associated with repressors of temperate phages. we have utilized these observations to demonstrate the use of l5 gene 71 as a selectable marker for genetic transformation o ... | 1993 | 8459767 |
| transcriptional regulation of repressor synthesis in mycobacteriophage l5. | mycobacteriophage l5 is a temperate phage of the mycobacteria that forms stable lysogens in mycobacterium smegmatis. lysogeny is maintained by the putative repressor, the gene 71 product, which also mediates immunity to superinfection. we show here that there are three promoters located upstream of gene 71 which are active in an l5 lysogen but which do not require any phage-encoded proteins. in early lytic growth, gene 71 is also transcribed from a promoter, pleft, located at the right end of th ... | 1995 | 8594325 |
| positions of strand exchange in mycobacteriophage l5 integration and characterization of the attb site. | mycobacteriophage l5 integrates into the genome of mycobacterium smegmatis via site-specific recombination between the phage attp site and the bacterial attb site. these two sites have a 43-bp common core sequence within which strand exchange occurs and which overlaps a trnagly gene at attb. we show here that a 29-bp segment of dna is necessary and sufficient for attb function and identify the positions of strand exchange. | 1996 | 8808947 |
| mycobacteriophage d29 contains an integration system similar to that of the temperate mycobacteriophage l5. | a mycobacteriophage d29 dna fragment cloned in prm64, a shuttle plasmid that transforms mycobacterium smegmatis, was sequenced. the determined sequence was 2592 nucleotides long and had a mean g+c content of 63.7 mol%, similar to that of mycobacterial dna. four orfs were identified: one with strong homology to dcmp deaminase genes; one homologous to mycobacteriophage l5 gene 36, whose function is unknown; one encoding a possible excisase; and one encoding an integrase. the intergenic region betw ... | 1997 | 9274023 |
| mycobacteriophage l5 infection of mycobacterium bovis bcg: implications for phage genetics in the slow-growing mycobacteria. | mycobacteriophage l5 is a well-characterized temperate phage that forms stable lysogens in mycobacterium smegmatis. the host range of l5 is, however, unclear because previous reports suggested that it does not infect slow-growing mycobacteria such as mycobacterium tuberculosis and bacille calmette-guerin (bcg). moreover, luciferase reporter phage derivatives of l5 failed to produce light from bcg, suggesting that infection is blocked at or before the stage of dna injection. in this study, we dem ... | 1997 | 9427405 |
| genome structure of mycobacteriophage d29: implications for phage evolution. | mycobacteriophage d29 is a lytic phage that infects both fast and slow-growing mycobacterial species. the complete genome sequence of d29 reveals that it is a close relative of the temperate mycobacteriophage l5, whose sequence has been described previously. the overall organization of the d29 genome is similar to that of l5, although a 3.6 kb deletion removing the repressor gene accounts for the inability of d29 to form lysogens. comparison of the two genomes shows that they are punctuated by a ... | 1998 | 9636706 |
| characterization of the mihf gene of mycobacterium smegmatis. | integration of mycobacteriophage l5 requires the mycobacterial integration host factor (mihf) in vitro. mihf is a 105-residue heat-stable polypeptide that is not obviously related to hu or any other small dna-binding proteins. mihf is most abundant just prior to entry into stationary phase and is essential for the viability of mycobacterium smegmatis. | 1998 | 9765584 |
| protein-dna complexes in mycobacteriophage l5 integrative recombination. | the temperate mycobacteriophage l5 integrates site specifically into the genomes of mycobacterium smegmatis, mycobacterium tuberculosis, and mycobacterium bovis bacillus calmette-guérin. this integrative recombination event occurs between the phage l5 attp site and the mycobacterial attb site and requires the phage-encoded integrase and mycobacterial-encoded integration host factor mihf. here we show that attp, int-l5, and mihf assemble into a recombinationally active complex, the intasome, whic ... | 1999 | 9882658 |
| mycobacteriophage d29 integrase-mediated recombination: specificity of mycobacteriophage integration. | mycobacteriophage d29 is a lytic phage that infects both fast- and slow-growing species of the mycobacteria. d29 forms clear plaques on lawns of mycobacterium smegmatis and mycobacterium bovis bacille calmette-guérin (bcg) in which a very high proportion of infected cells are killed. however, genomic analysis of d29 demonstrates that it is a close relative of the temperate mycobacteriophage l5, and is presumably a non-temperate derivative of a temperate parent. the d29 genome encodes a putative ... | 1998 | 9931474 |
| instability and site-specific excision of integration-proficient mycobacteriophage l5 plasmids: development of stably maintained integrative vectors. | integrative vectors expressing foreign genes are used as tools for the development of recombinant vaccines in mycobacteria since it is assumed that these vectors are stably maintained even without antibiotic selection. we here demonstrate that integration-proficient vectors are lost from the mycobacterial genome in high frequency. loss of integrated vectors occurred in reca+ and in reca-strains, indicating a reca-independent mechanism. loss of the integrated vector was prevented when integrase g ... | 2001 | 11310445 |
| expression of mycobacteriophage ms6 lysis genes is driven by two sigma(70)-like promoters and is dependent on a transcription termination signal present in the leader rna. | a mycobacteriophage ms6 strong promoter region (p(lys)) was isolated by using transcriptional fusions with the lacz reporter gene. two tandem sigma(70)-like promoter sequences (p1 and p2) were found in this region. dna sequencing of the promoter downstream region revealed a 214-bp leader sequence followed by five adjacent coding regions of 231 bp (orf1), 1,152 bp (orf2), 996 bp (orf3), 231 bp (orf4), and 372 (orf5). orf1 has the potential to encode a 77-amino-acid protein which revealed similari ... | 2002 | 12003945 |
| in silico analysis of mycobacteriophage che12 genome: characterization of genes required to lysogenise mycobacterium tuberculosis. | che12 is a temperate chennai phage infecting mycobacterium tuberculosis. the nucleotide sequence of the 52,047 bp linear double stranded dna genome has a gc content of 62.9% with 70 putative orfs identified. functions are assigned to 24 genes based on the similarity of the predicted products to known proteins. che12 genome is highly similar to mycobacteriophage l5 and d29 genomes. the overall genome similarity of che12 to l5 is 82.5% and d29 is 81.5%. the genes attributing to lysogeny such as in ... | 2007 | 17379577 |
| identification of three cytotoxic early proteins of mycobacteriophage l5 leading to growth inhibition in mycobacterium smegmatis. | mycobacteriophage l5 is a temperate phage with a broad host range among the fast- and slow-growing mycobacteria such as mycobacterium smegmatis, mycobacterium tuberculosis, mycobacterium avium and mycobacterium ulcerans. l5 switches off host protein synthesis during the early stage of lytic growth, as was previously shown by protein expression profiling. also, lethal genetic elements have been identified in l5 based on the fact that transformants could not be obtained with these genes. using an ... | 2008 | 18667563 |
| method to integrate multiple plasmids into the mycobacterial chromosome. | in order to create a system in which two independent plasmids can be integrated into a mycobacterial chromosome, a mycobacterial plasmid was constructed containing the phage attachment site attp from the mycobacteriophage l5 genome and additionally containing the bacterial attachment site, attb. this plasmid will integrate into the mycobacterial chromosome via recombination of the plasmid-borne attp site with the chromosomal attb site in the presence of a mycobacterial vector carrying the l5 int ... | 2004 | 14718555 |
| efficient switching of mycobacteriophage l5-based integrating plasmids in mycobacterium tuberculosis. | we previously used a mycobacteriophage l5-derived integrating vector to demonstrate that glne and arok are essential genes in mycobacterium tuberculosis by showing that we were unable to excise the integrated vector when it carried the only functional copy of these genes. we tested three systems to replace the integrated copy with alternative alleles. the most efficient method was to transform the strain with a second copy of the integrating vector. excision of the resident vector and integratio ... | 2003 | 14680701 |
| control of directionality in l5 integrase-mediated site-specific recombination. | mycobacteriophage l5 is a temperate phage that forms lysogens in mycobacterium smegmatis. these lysogens carry an integrated l5 prophage inserted at a specific chromosomal location and undergo subsequent excision during induction of lytic growth. both the integrative and excisive site-specific recombination events are catalyzed by the phage-encoded tyrosine integrase (int-l5) and require the host-encoded protein, mihf. the directionality of these recombination events is determined by a second ph ... | 2003 | 12581642 |
| use of the mycobacteriophage l5 excisionase in mycobacterium tuberculosis to demonstrate gene essentiality. | swtting: demonstrating that a gene is essential is always difficult, but this is particularly true for a slow-growing organism such as mycobacterium tuberculosis. one method currently used is to show that homologous recombination leading to gene inactivation only occurs in the presence of a second copy of the gene, but obtaining statistically significant data can be prohibitively difficult. l5-based integrating plasmids have been widely used in the genetic analysis of mycobacteria. the l5 excisi ... | 2001 | 11800587 |
| 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 simi ... | 2000 | 11123672 |
| assembly and activation of site-specific recombination complexes. | site-specific recombination is responsible for a broad range of biological phenomena, including dna inversion, resolution of transposition intermediates, and the integration and excision of bacteriophage genomes. integration of mycobacteriophage l5 is catalyzed by a phage-encoded integrase with recombination occurring between specific attachment sites on the phage and mycobacterial chromosomes (attp and attb, respectively). although some site-specific recombination systems simply involve binding ... | 2000 | 10869430 |
| identification and characterization of mycobacteriophage l5 excisionase. | the well-characterized mycobacteriophage l5 forms stable lysogens in mycobacterium smegmatis. establishment of lysogeny involves integration of the phage genome into the chromosome of its mycobacterial hosts through an integrase-mediated site-specific recombination event. as l5 lysogens spontaneously generate free phage particles, prophage excision must also occur, although an l5 excisionase gene had not been identified. we show here that l5 gene 36 encodes the phage excisionase and is a small, ... | 2000 | 10652095 |
| molecular cloning, sequencing, and expression of two late proteins of bacteriophage mb78. | bacteriophage mb78, a virulent phage of salmonella typhimurium, does not allow other phages, such as p22 and 9na, to grow in its presence. a detailed physical map of this phage has been constructed in our laboratory. in an ongoing effort to understand the genetics of this interesting phage, various genes were characterized. here, we report cloning, sequencing, and expression of two late proteins, coded in a sali-hindiii fragment (sh9), by using the minicell expression system. further, we perform ... | 1999 | 10637764 |
| exposure to antibiotics induces expression of the mycobacterium tuberculosis sigf gene: implications for chemotherapy against mycobacterial persistors. | the sigf gene encodes an alternate sigma factor found in mycobacterium tuberculosis and related pathogenic mycobacteria. determination of conditions of sigf expression is an important step in understanding the conditional gene regulation which may govern such processes as virulence and dormancy in mycobacteria. we constructed an in-frame translational lacz-kan fusion within the sigf gene to determine the conditions of sigf expression. this reporter construct was expressed from a multicopy plasmi ... | 1999 | 9925509 |
| the role of supercoiling in mycobacteriophage l5 integrative recombination. | the genome of temperate mycobacteriophage l5 integrates into the chromosomes of its hosts, including mycobacterium smegmatis , mycobacterium tuberculosis and bacille calmette-guérin. this integrase-mediated site-specific recombination reaction occurs between the phage attp site and the mycobacterial attb site and requires the mycobacterial integration host factor. here we examine the role of supercoiling in this reaction and show that integration is stimulated by dna supercoiling but that superc ... | 1998 | 9705513 |
| expression systems for study of mycobacterial gene regulation and development of recombinant bcg vaccines. | successful genetic engineering of mycobacteria is crucial for developing new approaches to combat tuberculosis as well as for dissecting out the molecular basis of pathogenesis of mycobacterium tuberculosis. we have constructed a mycobacterium-escherichia coli shuttle expression vector psd5. it carries a modular expression cassette which provides sites for cloning of promoters, a ribosome binding site (rbs) with an appropriately placed initiation codon and multiple cloning sites for cloning the ... | 1998 | 9618292 |
| transcriptional silencing by the mycobacteriophage l5 repressor. | the success of a temperate bacteriophage is dependent upon its ability to completely shut down expression of its lytic genes during lysogenic growth. mycobacteriophage l5 accomplishes this by an atypical phage repressor, gp71, which binds to multiple asymmetric dna sites. l5 gp71 regulates transcription initiation at an early lytic promoter, pleft, but also affects downstream gene expression at 'stoperator' sites in the phage genome. the l5 genome is replete with stoperator sites located within ... | 1997 | 9312049 |
| characterization of the mycobacteriophage l5 attachment site, attp. | lysogenization of mycobacteriophage l5 involves integration of the phage genome into the mycobacterium smegmatis chromosome. integration occurs by a site-specific recombination event between a phage attachment site, attp, and a bacterial attachment site, attb, which is catalyzed by the phage-encoded integrase protein. dnase i footprinting reveals that l5 integrase binds to two types of sites within attp which span an unexpectedly large region of 413 bp: seven arm-type sites (p1 to p7) each of wh ... | 1997 | 9054972 |
| a novel host factor for integration of mycobacteriophage l5. | bacterial integration host factors (ihfs) play central roles in the cellular processes of recombination, dna replication, transcription, and bacterial pathogenesis. we describe here a novel mycobacterial ihf (mihf) of mycobacterium smegmatis and mycobacterium tuberculosis that stimulates integration of mycobacteriophage l5. mihf is the product of a single gene and is unrelated at the sequence level to other integration host factors. by itself, mihf does not bind preferentially to attp dna, altho ... | 1996 | 8986825 |
| the bxb1 gp47 recombination directionality factor is required not only for prophage excision, but also for phage dna replication. | mycobacteriophage bxb1 encodes a serine-integrase that catalyzes both integrative and excisive site-specific recombination. however, excision requires a second phage-encoded protein, gp47, which serves as a recombination directionality factor (rdf). the viability of a bxb1 mutant containing an s153a substitution in gp47 that eliminates the rdf activity of bxb1 gp47 shows that excision is not required for bxb1 lytic growth. however, the inability to construct a δ47 deletion mutant of bxb1 suggest ... | 2011 | 22227494 |
| highly fluorescent gfpm 2+ -based genome integration-proficient promoter probe vector to study mycobacterium tuberculosis promoters in infected macrophages. | study of activity of cloned promoters in slow-growing mycobacterium tuberculosis during long-term growth conditions in vitro or inside macrophages, requires a genome-integration proficient promoter probe vector, which can be stably maintained even without antibiotics, carrying a substrate-independent, easily scorable and highly sensitive reporter gene. in order to meet this requirement, we constructed pakmn2, which contains mycobacterial codon-optimized gfp(m) (2+) gene, coding for gfp(m) (2+) o ... | 2012 | 21958386 |
| the cytotoxic early protein 77 of mycobacteriophage l5 interacts with msmeg_3532, an l-serine dehydratase of mycobacterium smegmatis. | mycobacteriophage l5 is a temperate phage infecting a broad range of mycobacterial species. upon induction of lytic growth, l5 rapidly switches off host protein synthesis. we have recently identified the mycobacteriophage l5 early protein gp77 as a host shut-off protein that acts growth inhibitory in the mycobacterial host when expressed through the corresponding phage promoter. here we present data showing that this purified phage protein of unknown function specifically binds to protein msmeg_ ... | 2011 | 21656815 |
| transfer, stable maintenance and expression of the mycolactone polyketide megasynthase mls genes in a recombination-impaired mycobacterium marinum. | the human pathogen mycobacterium ulcerans produces a polyketide metabolite called mycolactone with potent immunomodulatory activity. m. ulcerans strain agy99 has a 174 kb plasmid called pmum001 with three large genes (mlsa1, 51 kb; mlsa2, 7.2 kb; mlsb, 43 kb) that encode type i polyketide synthases (pks) required for the biosynthesis of mycolactone, as demonstrated by transposon mutagenesis. however, there have been no reports of transfer of the mls locus to another mycobacterium to demonstrate ... | 2009 | 19383681 |
| overexpression of a delayed early gene hlg1 of temperate mycobacteriophage l1 is lethal to both m. smegmatis and e. coli. | two genes of temperate mycobacteriophage l5, namely, gp63 and gp64, were hypothesized to be toxic to m. smegmatis. an identical l5 gp64 ortholog (designated hlg1) was cloned from homoimmune mycobacteriophage l1 and characterized at length here. as expected, hlg1 affected the growth of m. smegmatis when overexpressed from a resident plasmid. hlg1 (the protein encoded by hlg1) in fact caused growth retardation of m. smegmatis and the region encompassing its 57-114 c-terminal amino acid residues wa ... | 2008 | 18510866 |
| cloning, characterization and expression analysis of nucleotide metabolism-related genes of mycobacteriophage l5. | the genomes of mycobacteriophages of the l5 family, which includes the lytic phage d29, contain several genes putatively linked to nucleotide-metabolizing functions. two such genes, 48 and 50, encoding thymidylate synthase and ribonucleotide reductase (rnr), respectively, were overexpressed in escherichia coli and the recombinant proteins were biochemically characterized. it was established that gp50 was a class ii rnr having properties similar to that of the corresponding enzyme from lactobacil ... | 2008 | 18248423 |
| comparative genomic analysis of mycobacteriophage tweety: evolutionary insights and construction of compatible site-specific integration vectors for mycobacteria. | mycobacteriophage tweety is a newly isolated phage of mycobacterium smegmatis. it has a viral morphology with an isometric head and a long flexible tail, and forms turbid plaques from which stable lysogens can be isolated. the tweety genome is 58 692 bp in length, contains 109 protein-coding genes, and shows significant but interrupted nucleotide sequence similarity with the previously described mycobacteriophages llij, pmc and che8. however, overall the genome possesses mosaic architecture, wit ... | 2007 | 17660435 |