Transposition-Induced Structural Instability ofEscherichia coli–Mycobacteria Shuttle Vectors

Chawla, Mamta; Gupta, S. K.

doi:10.1006/plas.1998.1384

Cited by 19 publications

(10 citation statements)

References 11 publications

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“…However, the presence of both insertions and deletions were reported by other workers (Haeseleer, 1994 ;Garbe et al, 1994 ;Kumar et al, 1998 ;Chawla & Das Gupta, 1999 ;De Smet et al, 1999), which would suggest that the changes were transposon-induced in both mc#155 and M. vaccae. Studies are under way in our laboratory to define the precise nature of the observed structural modifications.…”

Section: Analysis Of Vector Stabilitymentioning

confidence: 78%

Comparative evaluation of Mycobacterium vaccae as a surrogate cloning host for use in the study of mycobacterial genetics

et al. 2002

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Mycobacterium vaccae represents an alternative mycobacterial cloning host that has been largely overlooked to date. The main reason for this may be the reported non-transformability of this species, specifically the so-called Stanford strain (NCTC 11659), with expression vectors that use kanamycin resistance as a selection method. However, this strain can be transformed using hygromycin resistance as an alternative selectable phenotype. The present study has shown that in contrast to previous reports, M. vaccae (ATCC 15483) is capable of being transformed with a range of vectors encoding kanamycin resistance as the selectable marker. Thereafter, the expression of the lacZ reporter gene in M. vaccae, Mycobacterium bovis BCG and Mycobacterium smegmatis mc 2 155 was evaluated using a range of characterized mycobacterial promoter sequences (hsp60, hsp70, PAN, 18kDa and 16S rRNA) cloned in the same promoter probe vector. In general, the promoters showed similar levels of activity in the three species, demonstrating that existing expression systems can readily be employed with M. vaccae (ATCC 15483). This was further confirmed by the observation that M. vaccae was capable of stable, in vitro expression of recombinant S1 subunit of pertussis toxin at levels equivalent to those obtained with BCG and M. smegmatis. Analysis of structural and functional stability of a range of vectors demonstrated that the incidence of instability noted for M. vaccae was lower than that recorded for M. smegmatis. Taken together, the results indicate that M. vaccae is an additional cloning host which may prove useful for specific aspects of mycobacterial biology and provide increased flexibility to the field of recombinant protein technology for mycobacteria.

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Section: Analysis Of Vector Stabilitymentioning

confidence: 78%

Comparative evaluation of Mycobacterium vaccae as a surrogate cloning host for use in the study of mycobacterial genetics

et al. 2002

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“…These features are not ideal for the creation of stable, extra-chromosomal genomic libraries. Rather, vectors for creation of such libraries should be free of such features while including strong bidirectional, host factor independent transcriptional terminators around the multiple cloning site, as well as a single origin of replication to promote plasmid stability (Chawla and Das Gupta, 1999;Godiska, 2004;Santamaria, 2000;Vilette et al, 1995). Moreover, to be useful in various bacterial species, such vectors must include a broad host range replicon, a variety of options for controlling transcription via constitutive, inducible, or native promoters that rely on genomic ribosomal binding sites and start codons, and a range of antibiotic resistance genes.…”

Section: Introductionmentioning

confidence: 99%

Broad host range vectors for stable genomic library construction

Lynch

Gill

2006

Biotech & Bioengineering

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We describe the construction of 36 stable vectors for genomic library construction in gram-negative species. These vectors contain the pBBR1 replicon that has been shown to stably replicate in every gram-negative species tested. The plasmids also contain bidirectional, rho-independent transcriptional terminators flanking the multiple cloning site, which allows for greater insert stability, and thus, greater genomic representation. Each vector varies in its antibiotic resistance cassette, mobilization function, and promoter used to express insert sequences. These vectors should prove useful in the screening of highly representational genomic libraries in a broad variety of gram-negative species.

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“…Deletion mutants were constructed by directly amplifying the desired region using primers mentioned in Table S2 in the supplemental material and cloning into the expression vector. The transformation of mycobacteria was performed by using the pAL5000-based E. coli-Mycobacterium shuttle vector pMC2 (13), using electroporation. For comparisons of transformation efficiencies, equal amounts of supercoiled wild-type and mutant pMC2 plasmid DNAs were used.…”

mentioning

confidence: 99%

Evolutionary Link between the Mycobacterial Plasmid pAL5000 Replication Protein RepB and the Extracytoplasmic Function Family of σ Factors

Basu

Chatterjee

et al. 2012

J Bacteriol

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Mycobacterial plasmid pAL5000 represents a family of plasmids found mostly in the Actinobacteria. It replicates using two plasmid-encoded proteins, RepA and RepB. While BLAST searches indicate that RepA is a replicase family protein, the evolutionary connection of RepB cannot be established, as no significant homologous partner (E < 10 ؊3 ) outside the RepB family can be identified. To obtain insight into the structure-function and evolutionary connections of RepB, an investigation was undertaken using homology modeling, phylogenetic, and mutational analysis methods. The results indicate that although they are synthesized from the same operon, the phylogenetic affinities of RepA and RepB differ. Thus, the operon may have evolved through random breaking and joining events. Homology modeling predicted the presence of a three-helical helix-turn-helix domain characteristic of region 4 of extracytoplasmic function (ECF) factors in the C-terminal region of RepB. At the N-terminal region, there is a helical stretch, which may be distantly related to region 3 of factors. Mutational analysis identified two arginines indispensable for RepB activity, one each located within the C-and N-terminal conserved regions. Apart from analyzing the domain organization of the protein, the significance of the presence of a highly conserved A/T-rich element within the RepB binding site was investigated. Mutational analysis revealed that although this motif does not bind RepB, its integrity is important for efficient DNA-protein interactions and replication to occur. The present investigation unravels the possibility that RepB-like proteins and their binding sites represent ancient DNA-protein interaction modules.

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Transposition-Induced Structural Instability ofEscherichia coli–Mycobacteria Shuttle Vectors

Cited by 19 publications

References 11 publications

Comparative evaluation of Mycobacterium vaccae as a surrogate cloning host for use in the study of mycobacterial genetics

Comparative evaluation of Mycobacterium vaccae as a surrogate cloning host for use in the study of mycobacterial genetics

Broad host range vectors for stable genomic library construction

Evolutionary Link between the Mycobacterial Plasmid pAL5000 Replication Protein RepB and the Extracytoplasmic Function Family of σ Factors

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