Genetic transformation of BCG

Lugosi, L; Jacobs, William R.; Bloom, Barry R.

doi:10.1016/0041-3879(89)90046-9

Cited by 31 publications

(5 citation statements)

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“…Conditions for transfection and transformation of M. avium complex and M. paratuberculosis were optimi2ed based on previous studies with M. smegmatis and BCG (Barletta et al, 1992; Jacobs et al, 1991; Lugosi et al, 1989; Snapper et al, 1988, 1990). Mycobacterial cells were grown in complete Middlebrook 7H9 medium as described above.…”

Section: Methodsmentioning

confidence: 99%

Phage infection, transfection and transformation of Mycobacterium avium complex and Mycobacterium paratuberculosis

et al. 1995

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Mycobacterium awium complex strains and Mycobacterium paratuberculosis are closely related intracellular pathogens affecting humans and animals. M. awium complex infections are a leading cause of morbidity and mortality in AIDS patients, and M. paratuberculosis is the agent of Johne's disease in ruminants. Genetic manipulation of these micro-organisms would facilitate the understanding of their pathogenesis, the construction of attenuated vaccine strains and the development of new drugs and treatment methods. This paper describes the replication of mycobacterial shuttle phasmids and plasmids, and the expression of the firefly luciferase reporter gene in M. awium complex and M. paratuberculosis. The mycobacteriophage TM4 propagated on M. smegmatis or M. paratuberculosis plaqued at the same efficiency on these two mycobacterial hosts. Screening of M. awium complex and M. paratubemulosis clinical isolates with T W e r i v e d luciferase reporter phages demonstrated that the majority of these isolates were susceptible t o TM4. Conditions for introduction of DNA were determined by transfection of M. paratuberculosis with TM4 DNA and applied t o isolate kanamycin-resistant transformants of M. a wium complex and M. paratubemulosir with ischerichia coli-Mycobacterium shuttle plasmids. Recombinant plasmids were recovered from transformanb without apparent loss of DNA sequences. These results provide the basis for the genetic manipulation of these pathogenic mycobacterial species. were grown standing at 37 OC in Middlebrook 7H9 broth, adjusted to pH 5.9, and supplemented with oleic acid/albumin/dextrose complex and 005 % Tween 80. Ferric mycobactin J (Allied Monitor) at 1.0 pg ml-' was added for M. paratuberculosis cultures. For solid media, Tween was omitted and Bacto Agar was added to 7H9 Middlebrook medium at 15 g 1-l. High-titre lysates were prepared and purified by caesium chloride equilibrium density centrifugation, as previously described (Jacobs et al., 1991). Mycobacteriophage infection assays.Phage lysates (0.1 ml of duplicate 10-fold serial dilutions) were incubated with 0.2 ml of fresh mycobacterial cultures, corresponding to approximately 3.0 x lo7 c.f.u., for 30 min at room temperature. Middlebrook 7H9 soft agar (0.7 %) was added, and the cells were plated on 7H9 Middlebrook medium by the soft agar layer method as described by Adams (1959) and incubated at 37 OC until either plaques or confluent lawns developed (1 to 3 d for M. smegmatis, 2 to 4 weeks for M. avium complex strains and M. paratuberculosis). Phage titres were determined at dilutions that gave single isolated plaques to exclude the possibility of lysis from without.Infection with luciferase reporter phasmids and luciferase assays. Infection with luciferase reporter phages and luciferase assays were performed as described by Jacobs et al. (1993). Mycobacterial cultures were grown to exponential phase (OD,,, 0-2; approximately 6.0 x lo7 c.f.u. ml-l) in Middlebrook 7H9 medium, washed three times in growth medium without Tween, 1984), was likely ...

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Section: Methodsmentioning

confidence: 99%

Phage infection, transfection and transformation of Mycobacterium avium complex and Mycobacterium paratuberculosis

et al. 1995

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“…M. smegmatis mc 2 155 was a generous gift from W. R. Jacobs, Albert Einstein College of Medicine, Bronx, NY (18) and was transformed as described previously (19). Recombinant clones were selected on Middlebrook 7H10 agar supplemented with oleic acid-albumin-dextrose-catalase enrichment (Difco, Detroit, MI) containing 25 g/ml kanamycin (Sigma).…”

Section: Bioinformatics-the Transmembrane Segments (Tms) Of Mtppm1mentioning

confidence: 99%

In Vivo Interaction between the Polyprenol Phosphate Mannose Synthase Ppm1 and the Integral Membrane Protein Ppm2 from Mycobacterium smegmatis Revealed by a Bacterial Two-hybrid System

Baulard¹,

Gurcha²,

Engohang‐Ndong³

et al. 2003

Journal of Biological Chemistry

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Dolichol phosphate-mannose (Dol-P-Man) is a mannose donor in various eukaryotic glycosylation processes. So far, two groups of Dol-P-Man synthases have been characterized based on the way they are stabilized in the endoplasmic reticulum membrane. Enzymes belonging to the first group, such as the yeast Dpm1, are typical integral membrane proteins harboring a transmembrane segment (TMS) at their C terminus. In contrast, mammalian Dpm1, enzymes of the second group, lack the typical TMS and require the association with the small hydrophobic proteins Dpm3 to be properly stabilized in the endoplasmic reticulum membrane. In Mycobacterium tuberculosis, the Polyprenol-P-Man synthase MtPpm1 is involved in the biosynthesis of the cell wall-associated glycolipid lipoarabinomannan. MtPpm1 is composed of two domains. The C-terminal catalytic domain is homologous to eukaryotic Dol-P-Man synthases. The Nterminal domain of MtPpm1 contains six TMS that anchor the enzyme in the cytoplasmic membrane. In contrast, in Mycobacterium smegmatis, orthologs of the two domains of MtPpm1 are encoded by two distinct open reading frames, Msppm1 and Msppm2, organized as an operon. No TMS are predicted in MsPpm1, and subcellular fractionation experiments indicate that this enzyme is cytosolic when produced in Escherichia coli. Computer-assisted topology predictions and alkaline phosphatase insertions showed that MsPpm2 is an integral membrane protein. Using a recently developed bacterial two-hybrid system, it was found that MsPpm2 interacts with MsPpm1 to stabilize the synthase MsPpm1 in the bacterial membrane. This interaction is reminiscent of that of mammalian Dpm1 with Dpm3 and mimics the structure of MtPpm1 as demonstrated by the capacity of the two domains of MtPpm1 to spontaneously interact when co-expressed in E. coli.Dolichol phosphate mannose (Dol-P-Man) 1 synthase transfers mannose (Man) from GDP-Man to polyisoprenoid dolichol phosphate (Dol-P). Dol-P-Man is a mannosyl donor in pathways leading to N-glycosylation (reviewed in Refs. 1 and 2), the synthesis of glycosylphosphatidylinositol anchors (3, 4), O-mannosylation of fungal proteins (4), and the construction of bacterial cell walls and protozoan glycocalyx (5, 6).The well characterized Dol-P-Man synthases fall into two distinct groups. One group contains the Schizosaccharomyces pombe, Caenorhabditis briggsiae, and mammalian Dol-P-Man synthases. The other group includes the Saccharomyces cerevisiae, Ustilago maydis, and Trypanosoma brucei Dol-P-Man synthases. Enzymes of the mammalian class lack the C-terminal hydrophobic domain that is characteristic of the S. cerevisiae class of Dol-P-Man synthases (7). The C-terminal hydrophobic domain has been proposed to stabilize the yeast enzyme by anchoring it in the cytoplasmic membrane. More recently, the mammalian Dol-P-Man synthase Dpm1 has been shown to interact with the transmembrane protein Dpm3 that retains and stabilizes the synthase in the endoplasmic reticulum membrane (8, 9), thereby functionally replacing the missing Cterminal ...

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“…M. smegmatis mc#155 was a gift from W. R. Jacobs (Albert Einstein College of Medicine, Bronx, New York, U.S.A.) [15]. M. smegmatis mc#155 was transformed as described previously [16], and recombinant clones were selected on Middlebrook 7H10 agar supplemented with oleic acid\albumin\dextrose\catalase enrichment (Difco, Detroit, MI, U.S.A.) containing 25 µg\ml kanamycin (Sigma). Liquid cultures of M. smegmatis (pMV261) and M. smegmatis (pMV261-Mt-ppm1) [also M. smegmatis (pMV261-Mt-ppm1\ D1) and M. smegmatis (pMV261-Mt-ppm1\D2)] were grown at 37 mC in Luria-Bertani broth (Difco) supplemented with 25 µg\ ml kanamycin and 0.05 % (v\v) Tween 80.…”

Section: Experimental Bacterial Strains and Growth Conditionsmentioning

confidence: 99%

Ppm1, a novel polyprenol monophosphomannose synthase from Mycobacterium tuberculosis

Gurcha

Baulard

Kremer

et al. 2002

Biochemical Journal

112

129

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Dolichol monophosphomannose (DPM) is an ever-present donor of mannose (Man) in various eukaryotic glycosylation processes. Intriguingly, the related polyprenol monophosphomannose (PPM) is involved in the biosynthesis of lipomannan and lipoarabinomanan, key bacterial factors termed modulins that are found in mycobacteria. Based on similarities to known DPM synthases, we have identified and characterized the PPM synthase of Mycobacterium tuberculosis, now termed Mt-Ppm1. In the present study, we demonstrate that Mt-Ppm1 possesses an unusual two-domain architecture, by which the second domain is sufficient for PPM synthesis. However, when overexpressed separately in mycobacteria, domain 1 of Mt-Ppm1 appears to increase the synthesis of PPM. Interestingly, other mycobacteria such as M. smegmatis, M. avium and M. leprae produce two distinct proteins, which are similar to the two domains found in Mt-Ppm1. Using an in vitro assay, we also demonstrate that Mt-Ppm1 transfers Man from GDP-Man to a structurally diverse range of lipid monophosphate acceptors. The identification of the PPM synthase as a key enzyme in lipoarabinomannan biosynthesis now provides an attractive candidate for gene disruption to generate mutants for subsequent immunological studies. PPM synthase can also be exploited as a target for specific inhibitors of M. tuberculosis.

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Genetic transformation of BCG

Cited by 31 publications

References 6 publications

Phage infection, transfection and transformation of Mycobacterium avium complex and Mycobacterium paratuberculosis

Phage infection, transfection and transformation of Mycobacterium avium complex and Mycobacterium paratuberculosis

In Vivo Interaction between the Polyprenol Phosphate Mannose Synthase Ppm1 and the Integral Membrane Protein Ppm2 from Mycobacterium smegmatis Revealed by a Bacterial Two-hybrid System

Ppm1, a novel polyprenol monophosphomannose synthase from Mycobacterium tuberculosis

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