The Molecular Genetics of Mycobacterium tuberculosis

Hatfull, Graham F.

doi:10.1007/978-3-642-80166-2_2

Cited by 13 publications

(11 citation statements)

References 47 publications

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“…Based on our previous experience in using conditionally replicating shuttle phasmid vectors (Hatfull, 1996 ;Hatfull et al, 1994 ;Jacobs et al, 1991) for efficient delivery of transposon constructs and generation of transposon libraries in both M. bovis BCG and M. tuberculosis (Bardarov et al, 1997 ;Cox et al, 1999) we sought the opportunity to extend their usage by S. Bardarov and others modifying them into in vitro-generated specialized transducing phages. Specialized transduction as a method for delivery of recombinant DNA into mycobacteria has several useful features.…”

Section: Discussionmentioning

confidence: 99%

Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis

et al. 2002

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The authors have developed a simple and highly efficient system for generating allelic exchanges in both fast-and slow-growing mycobacteria. In this procedure a gene of interest, disrupted by a selectable marker, is cloned into a conditionally replicating (temperature-sensitive) shuttle phasmid to generate a specialized transducing mycobacteriophage. The temperaturesensitive mutations in the mycobacteriophage genome permit replication at the permissive temperature of 30 SC but prevent replication at the nonpermissive temperature of 37 SC. Transduction at a non-permissive temperature results in highly efficient delivery of the recombination substrate to virtually all cells in the recipient population. The deletion mutations in the targeted genes are marked with antibiotic-resistance genes that are flanked by γδ-res (resolvase recognition target) sites. The transductants which have undergone a homologous recombination event can be conveniently selected on antibiotic-containing media. To demonstrate the utility of this genetic system seven different targeted gene disruptions were generated in three substrains of Mycobacterium bovis BCG, three strains of Mycobacterium tuberculosis, and Mycobacterium smegmatis. Mutants in the lysA, nadBC, panC, panCD, leuCD, Rv3291c and Rv0867c genes or operons were isolated as antibiotic-resistant (and in some cases auxotrophic) transductants. Using a plasmid encoding the γδ-resolvase (tnpR), the resistance genes could be removed, generating unmarked deletion mutations. It is concluded from the high frequency of allelic exchange events observed in this study that specialized transduction is a very efficient technique for genetic manipulation of mycobacteria and is a method of choice for constructing isogenic strains of M. tuberculosis, BCG or M. smegmatis which differ by defined mutations.

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Section: Discussionmentioning

confidence: 99%

Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis

et al. 2002

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“…Temperate phages have this potential as observed by the use of L5 gp71 selection for the construction of recombinant bacterial vaccines. [23][24] Description of both temperate and generalized transducing phages provides an opportunity to investigate the genetics of both phage as well as its host. 22 The approach using genomics of temperate phages of Staphylococcus aureus has been employed to identify phage ORFs with antimicrobial activity and their cognate bacterial targets for subsequent drug discovery.…”

Section: Discussionmentioning

confidence: 99%

Characterization of temperate phage Che12 and construction of a new tool for diagnosis of tuberculosis

et al. 2008

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SUMMARYA 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.

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“…Thus, some of the activated sulfate in these species is channeled into a separate pathway that leads to incorporation of sulfate into organic compounds. In this regard, it may be significant to note that M. tuberculosis produces sulfated glycolipids known as sulfatides (12). Thus, APS reductase may represent a key enzyme for the division of the reduction and sulfation pathways.…”

Section: Discussionmentioning

confidence: 99%

Identification of a New Class of 5′-Adenylylsulfate (APS) Reductases from Sulfate-Assimilating Bacteria

et al. 2000

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A gene was cloned from Burkholderia cepacia DBO1 that is homologous with Escherichia coli cysH encoding 3-phosphoadenylylsulfate (PAPS) reductase. The B. cepacia gene is the most recent addition to a growing list of cysH homologs from a diverse group of sulfate-assimilating bacteria whose products show greater homology to plant 5-adenylylsulfate (APS) reductase than they do to E. coli CysH. The evidence reported here shows that the cysH from one of the species, Pseudomonas aeruginosa, encodes APS reductase. It is able to complement an E. coli cysH mutant and a cysC mutant, indicating that the enzyme is able to bypass PAPS, synthesized by the cysC product. Insertional knockout mutation of P. aeruginosa cysH produced cysteine auxotrophy, indicating its role in sulfate assimilation. Purified P. aeruginosa CysH expressed as a His-tagged recombinant protein is able to reduce APS, but not PAPS. The enzyme has a specific activity of 5.8 mol ⅐ min ؊1 ⅐ mg of protein ؊1 at pH 8.5 and 30°C with thioredoxin supplied as an electron donor. APS reductase activity was detected in several bacterial species from which the novel type of cysH has been cloned, indicating that this enzyme may be widespread. Although an APS reductase from dissimilatory sulfate-reducing bacteria is known, it shows no structural or sequence homology with the assimilatory-type APS reductase reported here. The results suggest that the dissimilatory and assimilatory APS reductases evolved convergently.

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The Molecular Genetics of Mycobacterium tuberculosis

Cited by 13 publications

References 47 publications

Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis

Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis

Characterization of temperate phage Che12 and construction of a new tool for diagnosis of tuberculosis

Identification of a New Class of 5′-Adenylylsulfate (APS) Reductases from Sulfate-Assimilating Bacteria

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