Isolation and characterization of efficient plasmid transformation mutants of <i>Mycobacterium smegmatis</i>

Snapper, Scott B.; Melton, Rachel E.; Mustafa, Shuhaimi; Kieser, Tobias; Jacobs, William R.

doi:10.1111/j.1365-2958.1990.tb02040.x

Cited by 1,184 publications

(688 citation statements)

References 27 publications

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“…Generation of clarithromycin-resistant M. smegmatis mutants and identification of resistance-associated mutations Clarithromycin-resistant mutants of M. smegmatis mc 2 155 (Snapper et al, 1990) and of M. smegmatis mc 2 155 SMR5, a spontaneous streptomycin-resistant mutant of mc 2 155 (Sander et al, 1995), were obtained with a frequency of 0.7 × 10 ¹8 on LB medium containing clarithromycin (50 g ml ¹1 ). No major difference in the mutational rate was seen when plating M. smegmatis SMR5 rrnB ¹ or rrnA ¹ , genetically engineered strains of M. smegmatis mc 2 155 SMR5 containing a single functional rRNA (rrnA or rrnB ) operon (Sander et al, 1996b).…”

Section: Resultsmentioning

confidence: 99%

The role of ribosomal RNAs in macrolide resistance

Sander

Prammananan

Meier

et al. 1997

Molecular Microbiology

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SummaryMacrolides are bacteriostatic antibiotics which interfere with the peptidyltransfer function of the ribosome. We have investigated the molecular mechanisms underlying macrolide resistance in Mycobacterium smegmatis, an eubacterium carrying two rRNA operons. Surprisingly, drug resistance was associated not with alterations in ribosomal proteins, but with a single point mutation in the peptidyltransferase region of one of the two 23S RNA genes, i.e. A2058→G or A2059→G. This mutation resulted in a heterozygous organism with a mutated and a wild-type rRNA operon respectively. Reverse transcriptase sequencing indicated the expression of both wild-type and mutated rRNAs. The mutated operon was introduced into genetically engineered rrn ¹ strains of M. smegmatis carrying a single functional rRNA operon and into parental M. smegmatis with two chromosomal rRNA operons, using gene transfer as well as gene replacement techniques. The results obtained demonstrate the dominant nature of resistance. As exemplified in our results on macrolide resistance, a complete set of genetic tools is now available, which allows questions of dominance vs. recessivity and gene dosage effects in eubacterial ribosomal nucleic acids to be addressed experimentally in vivo.

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Section: Resultsmentioning

confidence: 99%

The role of ribosomal RNAs in macrolide resistance

Sander

Prammananan

Meier

et al. 1997

Molecular Microbiology

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“…E. coli cells were grown at 37ЊC in L-medium (Sambrook et al, 1989) containing the antibiotics, where appropriate, at the following concentrations: ampicillin and methicillin, 50 g ml ¹1 ; streptomycin, 100 g ml ¹1 ; kanamycin, 50 g ml ¹1 . M. smegmatis mc 2 155 (Snapper et al, 1990) was grown in Lemco or modified Dubos medium (Difco) at 37ЊC with the addition of 25 g kanamycin ml ¹1 for strains containing plasmids. The plasmids used in this study are listed in Table 1. Recombinant DNA techniques M. smegmatis DNA was isolated as described previously (Davis et al, 1991).…”

Section: Methodsmentioning

confidence: 99%

Mycobacterial recA is cotranscribed with a potential regulatory gene called recX

Kg¹,

Movahedzadeh²,

et al. 1997

Molecular Microbiology

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SummaryThe recA gene of Mycobacterium smegmatis has been cloned and sequenced. The amino acid sequence of the RecA protein is highly homologous to other RecA proteins. Three other potential open reading frames were identified. One of these showed extensive homology to a protein, HypB, involved in the incorporation of nickel into hydrogenases. Another, found downstream of and overlapping recA, was similar to a gene, recX, which has been proposed to play a regulatory role related to recA function. The homology between the M. smegmatis sequence and that of Mycobacterium tuberculosis extended upstream of the recA coding region for 140 bp including a motif identical to the Cheo-box consensus sequence which has been shown to bind LexA. In addition, the transcriptional start sites were found to be identical to those identified previously for M. tuberculosis. Transcriptional fusions to the reporter gene chloramphenicol acetyltransferase (CAT) revealed that recA was DNA-damage inducible and that expression required sequences at some distance from the mapped transcriptional start sites. Although a motif with only one mismatch to the Cheo box was found in the intergenic region between orf1 and orf2 these open reading frames were not DNA-damage inducible, nor was this motif required for regulation of recA expression. Gel retardation assays revealed that the reason for this was that LexA did not bind to this sequence containing a mismatch. Reverse transcription/polymerase chain reaction analysis of M. smegmatis RNA demonstrated that recA and orf3 (recX ) are within the same trancriptional unit.

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“…smegmatis [8,9] have permitted the expression of foreign genes in mycobacteria, including those from Myco. tuberculosis [10].…”

Section: Activation Of T Cells By Mycobacterial Proteins Is Central Tmentioning

confidence: 99%

Expression of Mycobacterium tuberculosis MPT64 in recombinant Myco. smegmatis: purification, immunogenicity and application to skin tests for tuberculosis

Roche

Winter

Triccas

et al. 1996

Clinical and Experimental Immunology

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SUMMARYProteins secreted across the cell wall of mycobacteria are important antigens recognized early in the host response to mycobacterial infection. MPT64 is a 23-kD secreted protein restricted to members of the Mycobacterium tuberculosis complex which elicits T cell responses and cutaneous DTH reactions in Myco. tuberculosis-infected animals. Patients with tuberculosis and their tuberculin-positive contacts respond to the protein, but recipients of bacille Calmette-Guérin (BCG) vaccine strains lacking the mpt64 gene do not. In the present study, we describe the development of a unique recombinant mycobacterial vector which secretes the encoded Myco. tuberculosis protein MPT64 at high levels into the culture filtrate, from which the protein is isolated by a single-step affinity chromatographic step. The purified protein was recognized by both polyclonal and monoclonal anti-MPT64 antibodies. The T cell reactivity of the protein was confirmed by its ability to stimulate human anti-rMPB64 T cell lines. The Myco. smegmatis recombinant MPT64 protein was superior to the Escherichia coli rMPB64 protein, which has identical amino acid sequence, in eliciting cutaneous DTH reactions in guinea pigs sensitized with Myco. tuberculosis. Animals sensitized with BCG strains lacking the mpb64 gene failed to respond to MPT64. Similarly, interferon-gamma (IFN-) responses in tuberculosis patients and their contacts were higher to the Myco. smegmatis form of the protein. The potential of this form of the Myco. tuberculosis MPT64 protein as a skin test reagent for tuberculosis is discussed.

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Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis

Cited by 1,184 publications

References 27 publications

The role of ribosomal RNAs in macrolide resistance

The role of ribosomal RNAs in macrolide resistance

Mycobacterial recA is cotranscribed with a potential regulatory gene called recX

Expression of Mycobacterium tuberculosis MPT64 in recombinant Myco. smegmatis: purification, immunogenicity and application to skin tests for tuberculosis

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