Resitriction endonuclease mapping and cloning of Mycobacterium fortuitum var. fortuitum plasmid pAL5000

Labidi, A; David, Hugo L.; Roulland-Dussoix, Daisy

doi:10.1016/s0769-2609(85)80045-4

Cited by 58 publications

(27 citation statements)

References 15 publications

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“…Cryptic plasmids have been found in members of the M. fortuitum complex (304). One of those M. fortuitum plasmids was used to construct a useful mycobacterial cloning vector (305), and methods for transformation of plasmid DNA into mycobacterial cells have been developed (251).…”

Section: Genetics Of Nontuberculous Mycobacteriamentioning

confidence: 99%

Epidemiology of Infection by Nontuberculous Mycobacteria

Martin

Parker²,

Falkinham

1987

Am Rev Respir Dis

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Section: Genetics Of Nontuberculous Mycobacteriamentioning

confidence: 99%

Epidemiology of Infection by Nontuberculous Mycobacteria

Martin

Parker²,

Falkinham

1987

Am Rev Respir Dis

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“…The isolation of plasmids which replicate in the mycobacteria, for example pAL5000 from Mycobacterium fortuitum (Labidi et al, 1985), has facilitated the construction of vectors for studying mycobacterial gene expression in a homologous host (Snapper et al, 1988 ;Aldovini & Young, 1991 ;Stover et al, 1991 ;Barletta et al, 1992). The surrogate host of choice is Mycobacterium smegmatis because of its non-pathogenicity and rapid growth.…”

Section: Introductionmentioning

confidence: 99%

The Mycobacterium tuberculosis katG promoter region contains a novel upstream activator

1999

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An Escherichia coli-mycobacterial shuttle vector, pJCluc, containing a luciferase reporter gene, was constructed and used to analyse the Mycobacterium tuberculosis katG promoter. A 19 kb region immediately upstream of katG promoted expression of the luciferase gene in E. coli and Mycobacterium smegmatis . A smaller promoter fragment (559 bp) promoted expression with equal efficiency, and was used in all further studies. Two transcription start sites were mapped by primer extension analysis to 47 and 56 bp upstream of the GTG initiation codon. Putative promoters associated with these show similarity to previously identified mycobacterial promoters. Deletions in the promoter fragment, introduced with BAL-31 nuclease and restriction endonucleases, revealed that a region between 559 and 448 bp upstream of the translation initiation codon, designated the upstream activator region (UAR), is essential for promoter activity in E. coli, and is required for optimal activity in M. smegmatis . The katG UAR was also able to increase expression from the Mycobacterium paratuberculosis P AN promoter 15-fold in E. coli and 12-fold in M. smegmatis . An alternative promoter is active in deletion constructs in which either the UAR or the katG promoters identified here are absent. Expression from the katG promoter peaks during late exponential phase, and declines during stationary phase. The promoter is induced by ascorbic acid, and is repressed by oxygen limitation and growth at elevated temperatures. The promoter constructs exhibited similar activities in Mycobacterium bovis BCG as they did in M. smegmatis .

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“…Subsequently these libraries were used to perform complementation of Escherichia coli mutants to identify mycobacterial genes (Garbe et al, 1990;Aldovini et al, 1993;Anderson and Hansen, 1993;Cirillo et al, 1994). Isolation of high-efficiency-transformation strains of the fast growing M. smegmatis (Snapper et al, 1990) and development of electroporation to transform mycobacteria (Snapper et al, 1988) were crucial achievements in our ability to express genes in mycobacteria with a variety of shuttle vectors that were based on the plasmid pAL5000 from M. fortuitum (Labidi et al, 1985;Snapper et al, 1988;Ranes et al, 1990;Stover et al, 1991). Plasmids and integration proficient vectors based on sequences derived from mycobacteriophages have enabled stable introduction and expression of genes in mycobacteria (Jacobs et al, 1987;Lee et al, 1991;Stover et al, 1991).…”

Section: Introductionmentioning

confidence: 99%

Construction of shuttle vectors for genetic manipulation and molecular analysis of mycobacteria

Jain

Kaushal

Dasgupta

et al. 1997

Gene

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Two novel shuttle vectors for mycobacteria are described which have been derived from the expression system pSD5 developed in our laboratory. Plasmid pSD5B is a promoter-selection vector containing a promoterless lacZ gene and allows the identification of mycobacterial promoters by the blue colour of the colonies on solid media containing XGal. Moreover, the chronological order of appearance of blue colonies and intensity of colour provide a qualitative index of transcriptional strengths of the cloned promoters. Plasmid pSD5C has been designed to construct mycobacterial genomic libraries and express the cloned DNA inserts as fusion proteins with maltose binding protein in mycobacteria. Libraries in pSD5C provide feasibility for their screening with either DNA probes or specific antisera for identifying the genes of interest and for isolation of specific genetic loci by complementation of Escherichia coli and mycobacterial mutants. These vectors combine the ease of working in E. coli with the advantage of directly propagating them in mycobacteria without further manipulations. Finally, we demonstrate that these vectors function efficiently both in fast growing Mycobacterium smegmatis and slow growing mycobacteria including Mycobacterium tuberculosis and Mycobacterium bovis BCG.

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Resitriction endonuclease mapping and cloning of Mycobacterium fortuitum var. fortuitum plasmid pAL5000

Cited by 58 publications

References 15 publications

Epidemiology of Infection by Nontuberculous Mycobacteria

Epidemiology of Infection by Nontuberculous Mycobacteria

The Mycobacterium tuberculosis katG promoter region contains a novel upstream activator

Construction of shuttle vectors for genetic manipulation and molecular analysis of mycobacteria

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