<i>Mycobacterium smegmatis</i> RNA polymerase: DNA supercoiling, action of rifampicin and mechanism of rifampicin resistance

Levin, Margaret; Hatfull, Graham F.

doi:10.1111/j.1365-2958.1993.tb01572.x

Cited by 95 publications

(70 citation statements)

References 28 publications

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“…The experiment was therefore performed using RNA isolated from M. smegmatis cells harbouring various katG promoter clones. The determination of transcription start sites for M. tuberculosis genes, using this heterologous host, has been shown to correlate well with results obtained from native mRNA transcripts (Levin & Hatfull, 1993 ;Dhandayuthapani et al, 1997 ;Movahedzadeh et al, 1997), and has successfully been used as a substitute in many cases (Murray et al, 1992 ;Kremer et al, 1995 ;Nesbit et al, 1995).…”

Section: Discussionsupporting

confidence: 54%

“…The surrogate host of choice is Mycobacterium smegmatis because of its non-pathogenicity and rapid growth. The M. smegmatis RNA polymerase has been purified and used for transcription initiation in vitro (Levin & Hatfull, 1993). The major form of this enzyme has marked conservation with that of E. coli (Predich et al, 1995), which suggests some common mechanisms of transcription initiation.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Discussionsupporting

confidence: 54%

Section: Introductionmentioning

confidence: 99%

The Mycobacterium tuberculosis katG promoter region contains a novel upstream activator

1999

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“…According to evaluation based on CLSI standards for slow-growing bacteria, these two isolates should be considered intermediately susceptible. On the other hand, in this study we have characterized the whole rpoB gene, recognized to be the RIF antibiotic target in prokaryotes (16,17,19), of all our isolates, and these two isolates carried only the B. melitensis biovar 3 molecular marker (M1249I). We can, therefore, assert that all isolates investigated were found to be sensitive to RIF and, in general, to all tested antimicrobial agents.…”

Section: Discussionmentioning

confidence: 99%

Molecular Epidemiological and Antibiotic Susceptibility Characterization of Brucella Isolates from Humans in Sicily, Italy

et al. 2007

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Brucellosis is a serious problem in Sicily. Brucella melitensis was identified as the species most frequently isolated in humans in Italy. No data, however, are available about the molecular epidemiological characterization of Brucella isolates from humans. We have conducted this study to molecularly characterize clinical isolates of Brucella spp. and to evaluate their antimicrobial susceptibilities. Twenty Brucella isolates were studied. Differential growth characteristics and DNA polymorphisms such as the restriction patterns of the PCR-amplified omp2a and omp2b genes, rpoB nucleotide sequencing, and multiple-locus variable-number tandem repeat analysis of 16 loci (MLVA-16) were used to characterize the strains. In vitro antibiotic susceptibility was determined by the E-test method on two different agar media, and the results were compared. All isolates were identified as B. melitensis biovar 3. rpoB nucleotide sequence analysis allowed the identification of two different genotypes of B. melitensis biovar 3. On the other hand, the MLVA-16 typing assay recognized 17 distinct genotypes. All isolates were sensitive to all tested antibiotics (rifampin, doxycycline, ciprofloxacin, ceftriaxone, and trimethoprim-sulfamethoxazole), and the Mueller-Hinton agar plate is recommended for antibiotic susceptibility testing by the E-test method. Our findings identify B. melitensis biovar 3 as the etiological agent isolated in Sicily and encourage the use of both molecular methods, and in particular of the MLVA-16 assay, in epidemiological trace-back analysis. This study represents the first epidemiological data from molecular typing of Brucella strains circulating in Italy and, in particular, in eastern Sicily.Brucellosis is an important health problem affecting animals and humans in many countries in the world and especially in the Mediterranean areas. Brucella infections may cause tremendous economic losses through reproductive failure in animals. The disease is transmitted by humans through the consumption of contaminated foods or by direct/indirect contact with infected animals. Brucella melitensis is the most important zoonotic agent, followed by Brucella abortus and Brucella suis.

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“…Because rifampin resistance-conferring mutations in rpoB have also been observed in nontuberculous Mycobacteria species (Honoré and Cole 1993;Levin and Hatfull 1993;Musser 1995) and because the use of 16S genotyping has proven problematic in speciating some mycobacteria (Fox et al 1992), the rpoB oligonucleotide array and conventional dideoxynucleotide sequencing were used to analyze sequence diversity within and between members of the mycobacterial genus. Among the 10 Mycobacterium species studied, analysis of the 705 nucleotides of the rpoB gene revealed that intraspecies variation was smallest within M. tuberculosis, M. xenopi, and M. kansasii species and greatest within M. gordonae, M. fortuitum, and M. smegmatis species.…”

Section: Discussionmentioning

confidence: 99%

Simultaneous Genotyping and Species Identification Using Hybridization Pattern Recognition Analysis of Generic Mycobacterium DNA Arrays

Gingeras¹,

Ghandour²,

Wang³

et al. 1998

Genome Res.

229

141

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High-density oligonucleotide arrays can be used to rapidly examine large amounts of DNA sequence in a high throughput manner. An array designed to determine the specific nucleotide sequence of 705 bp of the rpoB gene of Mycobacterium tuberculosis accurately detected rifampin resistance associated with mutations of 44 clinical isolates of M. tuberculosis. The nucleotide sequence diversity in 121 Mycobacterial isolates (comprised of 10 species) was examined by both conventional dideoxynucleotide sequencing of the rpoB and 16S genes and by analysis of the rpoB oligonucleotide array hybridization patterns. Species identification for each of the isolates was similar irrespective of whether 16S sequence, rpoB sequence, or the pattern of rpoB hybridization was used. However, for several species, the number of alleles in the 16S and rpoB gene sequences provided discordant estimates of the genetic diversity within a species. In addition to confirming the array's intended utility for sequencing the region of M. tuberculosis that confers rifampin resistance, this work demonstrates that this array can identify the species of nontuberculous Mycobacteria. This demonstrates the general point that DNA microarrays that sequence important genomic regions (such as drug resistance or pathogenicity islands) can simultaneously identify species and provide some insight into the organism's population structure.[The sequence data described in this paper have been submitted to GenBank under accession nos. AF09766-AF059853 and AF060279-AF060367.]For patients infected with Mycobacteria, especially those coinfected with the human immunodeficiency virus type 1 and type 2 (HIV-1, HIV-2), the identity of the Mycobacterium species and the presence of mutations that confer both biologically and clinically important phenotypes are of critical importance. Both of these issues have implications for the appropriate care and treatment of the infected patient. For example, although M. avium complex (MAC) is the most common cause for both disseminated Mycobacterium disease and death in patients with AIDS in the developed world (∼25%-50% of adults and 10% of children with AIDS are infected [Inderlied et al. 1993], Mycobacterium tuberculosis infections are also found in these patient populations. Important public health and patient management decisions (e.g., the need for clinical isolation and the choice of the appropriate therapeutic regimen) depend on a timely and accurate identification of the infecting agent. (Moore et al. 1997).On the basis of the insights provided by previously characterized RIF resistant mutants in Escherichia coli (Ovchinnikov et al. 1983; Jin and Gross 7 Corresponding author. E-MAIL tom gingeras@affymetrix.com; FAX (408) 481-0422.

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Mycobacterium smegmatis RNA polymerase: DNA supercoiling, action of rifampicin and mechanism of rifampicin resistance

Cited by 95 publications

References 28 publications

The Mycobacterium tuberculosis katG promoter region contains a novel upstream activator

The Mycobacterium tuberculosis katG promoter region contains a novel upstream activator

Molecular Epidemiological and Antibiotic Susceptibility Characterization of Brucella Isolates from Humans in Sicily, Italy

Simultaneous Genotyping and Species Identification Using Hybridization Pattern Recognition Analysis of Generic Mycobacterium DNA Arrays

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