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1.
mBio ; 5(3): e01245-14, 2014 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-24895308

RESUMEN

UNLABELLED: Specialized transduction has proven to be useful for generating deletion mutants in most mycobacteria, including virulent Mycobacterium tuberculosis. We have improved this system by developing (i) a single-step strategy for the construction of allelic exchange substrates (AES), (ii) a temperature-sensitive shuttle phasmid with a greater cloning capacity than phAE87, and (iii) bacteriophage-mediated transient expression of site-specific recombinase to precisely excise antibiotic markers. The methods ameliorate rate-limiting steps in strain construction in these difficult-to-manipulate bacteria. The new methods for strain construction were demonstrated to generalize to all classes of genes and chromosomal loci by generating more than 100 targeted single- or multiple-deletion substitutions. These improved methods pave the way for the generation of a complete ordered library of M. tuberculosis null strains, where each strain is deleted for a single defined open reading frame in M. tuberculosis. IMPORTANCE: This work reports major advances in the methods of genetics applicable to all mycobacteria, including but not limited to virulent M. tuberculosis, which would facilitate comparative genomics to identify drug targets, genetic validation of proposed pathways, and development of an effective vaccine. This study presents all the new methods developed and the improvements to existing methods in an integrated way. The work presented in this study could increase the pace of mycobacterial genetics significantly and will immediately be of wide use. These new methods are transformative and allow for the undertaking of construction of what has been one of the most fruitful resources in model systems: a comprehensive, ordered library set of the strains, each of which is deleted for a single defined open reading frame.


Asunto(s)
Eliminación de Gen , Mycobacterium tuberculosis/genética , Recombinación Genética , Transducción Genética , Alelos , Secuencia de Bases , Expresión Génica , Orden Génico , Recombinación Homóloga , Humanos , Datos de Secuencia Molecular , Micobacteriófagos/fisiología , Mycobacterium tuberculosis/virología , Plásmidos/genética
2.
J Clin Microbiol ; 50(4): 1362-9, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22278833

RESUMEN

The difficulty of diagnosing active tuberculosis (TB) and lack of rapid drug susceptibility testing (DST) at the point of care remain critical obstacles to TB control. This report describes a high-intensity mycobacterium-specific-fluorophage (φ(2)GFP10) that for the first time allows direct visualization of Mycobacterium tuberculosis in clinical sputum samples. Engineered features distinguishing φ(2)GFP10 from previous reporter phages include an improved vector backbone with increased cloning capacity and superior expression of fluorescent reporter genes through use of an efficient phage promoter. φ(2)GFP10 produces a 100-fold increase in fluorescence per cell compared to existing reporter phages. DST for isoniazid and oxofloxacin, carried out in cultured samples, was complete within 36 h. Use of φ(2)GFP10 detected M. tuberculosis in clinical sputum samples collected from TB patients. DST for rifampin and kanamycin from sputum samples yielded results after 12 h of incubation with φ(2)GFP10. Fluorophage φ(2)GFP10 has potential for clinical development as a rapid, sensitive, and inexpensive point-of-care diagnostic tool for M. tuberculosis infection and for rapid DST.


Asunto(s)
Bacteriófagos/genética , Mycobacterium tuberculosis/metabolismo , Esputo/microbiología , Tuberculosis Pulmonar/diagnóstico , Antituberculosos/farmacología , Genes Reporteros , Vectores Genéticos , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Humanos , Isoniazida/farmacología , Kanamicina/farmacología , Pruebas de Sensibilidad Microbiana/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/virología , Ofloxacino/farmacología , Regiones Promotoras Genéticas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Rifampin/farmacología , Relación Señal-Ruido , Tuberculosis Pulmonar/microbiología
3.
Microbiology (Reading) ; 155(Pt 12): 4050-4057, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19744987

RESUMEN

Mycobacteriophages have played an important role in the development of genetic tools and diagnostics for pathogenic mycobacteria, including Mycobacterium tuberculosis. However, despite the isolation of numerous phages that infect mycobacteria, the mechanisms of mycobacteriophage infection remain poorly understood, and knowledge about phage receptors is minimal. In an effort to identify the receptor for phage I3, we screened a library of Mycobacterium smegmatis transposon mutants for phage-resistant strains. All four phage I3-resistant mutants isolated were found to have transposon insertions in genes located in a cluster involved in the biosynthesis of the cell-wall-associated glycopeptidolipid (GPL), and consequently the mutants did not synthesize GPLs. The loss of GPLs correlated specifically with phage I3 resistance, as all mutants retained sensitivity to two other mycobacteriophages: D29 and Bxz1. In order to define the minimal receptor for phage I3, we then tested the phage sensitivity of previously described GPL-deficient mutants of M. smegmatis that accumulate biosynthesis intermediates of GPLs. The results indicated that, while the removal of most sugar residues from the fatty acyl tetrapeptide (FATP) core of GPL did not affect sensitivity to phage I3, a single methylated rhamnose, transferred by the rhamnosyltransferase Gtf2 to the FATP core, was critical for phage binding.


Asunto(s)
Glucolípidos/biosíntesis , Glicopéptidos/biosíntesis , Micobacteriófagos/patogenicidad , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/virología , Secuencia de Bases , Elementos Transponibles de ADN/genética , ADN Bacteriano/genética , ADN Viral/genética , Genes Bacterianos , Glucolípidos/química , Glicopéptidos/química , Familia de Multigenes , Mutagénesis Insercional , Micobacteriófagos/genética , Mycobacterium smegmatis/genética , Receptores Virales/metabolismo
4.
Tuberculosis (Edinb) ; 88(6): 616-23, 2008 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-18511339

RESUMEN

A temperate phage, Che12, able to infect Mycobacterium tuberculosis, was isolated from soil samples taken from tuberculosis sanatorium area in Chennai, India. The plaque morphology of this phage showed varying grades of turbidity on lawns of M. tuberculosis. The temperate nature of Che12 was established by super infection immunity. Phage integration into the host genomic DNA was confirmed by Southern hybridization using Che12 DNA as a probe. PCR amplification and sequencing of a part of the integrated phage genome in a M. tuberculosis lysogen also confirmed the temperate nature of Che12. The morphology of the phage particles was observed by electron microscopy, revealing similarities to other mycobacteriophages like L5, D29 and TM4. A luciferase reporter phage, phAETRC16, was constructed by cloning firefly luciferase gene into Che12. Infection of viable M. tuberculosis cells by phAETRC16 resulted in expression of luciferase leading to sustained light output. Che12, a true temperate phage infecting M. tuberculosis, is thus ideally suited for developing a diagnostic tool facilitating rapid diagnosis of M. tuberculosis.


Asunto(s)
Micobacteriófagos/genética , Mycobacterium tuberculosis/virología , Tuberculosis Pulmonar/diagnóstico , Animales , ADN Viral/genética , Genes Reporteros , Humanos , India , Luciferasas de Luciérnaga/genética , Luciferasas de Luciérnaga/metabolismo , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Micobacteriófagos/aislamiento & purificación , Micobacteriófagos/ultraestructura , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/virología , Sobreinfección/inmunología , Tuberculosis Pulmonar/genética , Ensayo de Placa Viral
5.
J Bacteriol ; 189(15): 5495-503, 2007 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17526710

RESUMEN

Phosphate import is required for the growth of mycobacteria and is regulated by environmental inorganic phosphate (P(i)) concentrations, although the mechanism of this regulation has not been characterized. The expression of genes involved in P(i) acquisition is frequently regulated by two-component regulatory systems (2CRs) consisting of a sensor histidine kinase and a DNA-binding response regulator. In this work, we have identified the senX3-regX3 2CR as a P(i)-dependent regulator of genes involved in phosphate acquisition in Mycobacterium smegmatis. Characterization of senX3 mutants with different PhoA phenotypes suggests a dual role for SenX3 as a phosphatase or a phosphodonor for the response regulator RegX3, depending upon P(i) availability. Expression of PhoA activity required phosphorylation of RegX3, consistent with a role for phosphorylated RegX3 (RegX3 approximately P) as a transcriptional activator of phoA. Furthermore, purified RegX3 approximately P bound to promoter sequences from phoA, senX3, and the high-affinity phosphate transporter component pstS, demonstrating direct transcriptional control of all three genes. DNase I footprinting and primer extension analyses have further defined the DNA-binding region and transcriptional start site within the phoA promoter. A DNA motif consisting of an inverted repeat was identified in each of the promoters bound by RegX3 approximately P. Based upon our findings, we propose a model for P(i)-regulated gene expression mediated by SenX3-RegX3 in mycobacteria.


Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Mycobacterium smegmatis/metabolismo , Fosfatos/metabolismo , Fosfotransferasas/metabolismo , Proteínas Bacterianas/genética , Secuencia de Bases , Huella de ADN , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Eliminación de Gen , Prueba de Complementación Genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación Missense , Mycobacterium smegmatis/genética , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Fosfotransferasas/genética , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Sitio de Iniciación de la Transcripción
6.
FEMS Microbiol Lett ; 241(2): 271-6, 2004 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-15598543

RESUMEN

We have isolated and characterized a new generalized transducing phage, Bxz1, from soil sampling at a neighboring Wildlife Preservation Park. The hosts of the phage, measured by the formation of plaques, include fast growing Mycobacterium smegmatis and Mycobacterium vaccae. Bxz1 is capable of transducing chromosomal markers, point mutations, and plasmids at frequencies ranging from 10(-8) to 10(-6) per plaque forming unit between strains of M. smegmatis. We also demonstrated cotransduction of a transposon insertion linked to a point mutation of the ndh gene.


Asunto(s)
Micobacteriófagos/genética , Micobacteriófagos/aislamiento & purificación , Mycobacterium smegmatis/virología , Mycobacterium/virología , Transducción Genética , Conjugación Genética , Marcadores Genéticos , Micobacteriófagos/fisiología , Mycobacterium/genética , Mycobacterium smegmatis/genética , Plásmidos , Microbiología del Suelo
7.
J Bacteriol ; 185(16): 4983-91, 2003 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-12897018

RESUMEN

Although alkaline phosphatases are common in a wide variety of bacteria, there has been no prior evidence for alkaline phosphatases in Mycobacterium smegmatis. Here we report that transposon insertions in the pst operon, encoding homologues of an inorganic phosphate transporter, leads to constitutive expression of a protein with alkaline phosphatase activity. DNA sequence analysis revealed that M. smegmatis does indeed have a phoA gene that shows high homology to other phoA genes. The M. smegmatis phoA gene was shown to be induced by phosphate starvation and thus negatively regulated by the pst operon. Interestingly, the putative M. smegmatis PhoA has a hydrophobic N-terminal domain which resembles a lipoprotein signal sequence. The M. smegmatis PhoA was demonstrated to be an exported protein associated with the cell surface. Furthermore, immunoprecipitation of PhoA from [(14)C]acetate-labeled M. smegmatis cell lysates demonstrated that this phosphatase is a lipoprotein.


Asunto(s)
Fosfatasa Alcalina/química , Membrana Celular/metabolismo , Regulación Bacteriana de la Expresión Génica , Lipoproteínas/química , Mycobacterium smegmatis/enzimología , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Elementos Transponibles de ADN , Lipoproteínas/genética , Lipoproteínas/metabolismo , Datos de Secuencia Molecular , Mutagénesis Insercional , Mycobacterium smegmatis/genética , Operón , Fosfatos/metabolismo , Análisis de Secuencia de ADN
8.
Cell ; 113(2): 171-82, 2003 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-12705866

RESUMEN

Bacteriophages are the most abundant organisms in the biosphere and play major roles in the ecological balance of microbial life. The genomic sequences of ten newly isolated mycobacteriophages suggest that the bacteriophage population as a whole is amazingly diverse and may represent the largest unexplored reservoir of sequence information in the biosphere. Genomic comparison of these mycobacteriophages contributes to our understanding of the mechanisms of viral evolution and provides compelling evidence for the role of illegitimate recombination in horizontal genetic exchange. The promiscuity of these recombination events results in the inclusion of many unexpected genes including those implicated in mycobacterial latency, the cellular and immune responses to mycobacterial infections, and autoimmune diseases such as human lupus. While the role of phages as vehicles of toxin genes is well established, these observations suggest a much broader involvement of phages in bacterial virulence and the host response to bacterial infections.


Asunto(s)
Regulación Viral de la Expresión Génica/genética , Genoma Viral , Interacciones Huésped-Parásitos/genética , Mosaicismo/genética , Micobacteriófagos/genética , Mycobacterium/virología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Infecciones Bacterianas/genética , Infecciones Bacterianas/inmunología , Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/genética , ADN Viral/genética , Evolución Molecular , Regulación Bacteriana de la Expresión Génica/genética , Humanos , Microscopía Electrónica , Datos de Secuencia Molecular , Micobacteriófagos/metabolismo , Micobacteriófagos/ultraestructura , Mycobacterium/genética , Mycobacterium/patogenicidad , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/virología , Filogenia , Homología de Secuencia de Ácido Nucleico , Transducción de Señal/genética
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