Genetic Mapping of Antibiotic Resistance in Markers
            <i>Bacillus subtilis</i>

Goldthwaite, Claire; Dubnau, David; Smith, Issar

doi:10.1073/pnas.65.1.96

Cited by 65 publications

(47 citation statements)

References 16 publications

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“…IS169 is a B. subtilis 168 strain carrying a mutation (tsp-6) that confers resistance to thiostrepton (26). The allele, originally isolated as a spontaneous bryamycin resistance mutation and called bry-2 (12), was renamed tsp-6 for uniformity of nomenclature (26). B. subtilis strains carrying the tsp-6 allele are missing ribosomal protein L11 (38).…”

Section: Methodsmentioning

confidence: 99%

Loss of Ribosomal Protein L11 Blocks Stress Activation of the Bacillus subtilis Transcription Factor ς ^B

2001

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general stress response sigma factor of Bacillus subtilis, is activated when the cell's energy levels decline or the bacterium is exposed to environmental stress (e.g., heat shock, ethanol). Physical stress activates B through a collection of regulatory kinases and phosphatases (the Rsb proteins) which catalyze the release of B from an anti-B factor inhibitor. The means by which diverse stresses communicate with the Rsb proteins is unknown; however, a role for the ribosome in this process was suggested when several of the upstream members of the B stress activation cascade (RsbR, -S, and -T) were found to cofractionate with ribosomes in crude B. subtilis extracts. We now present evidence for the involvement of a ribosome-mediated process in the stress activation of B . B. subtilis strains resistant to the antibiotic thiostrepton, due to the loss of ribosomal protein L11 (RplK), were found to be blocked in the stress activation of B . Neither the energy-responsive activation of B nor stress-dependent chaperone gene induction (a B -independent stress response) was inhibited by the loss of L11. The Rsb proteins required for stress activation of B are shown to be active in the RplK ؊ strain but fail to be triggered by stress. The data demonstrate that the B. subtilis ribosomes provide an essential input for the stress activation of B and suggest that the ribosomes may themselves be the sensors for stress in this system.

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Section: Methodsmentioning

confidence: 99%

Loss of Ribosomal Protein L11 Blocks Stress Activation of the Bacillus subtilis Transcription Factor ς ^B

2001

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“…A mutation in the lmr promoter region (lin-2) that confers lincomycin resistance in B. subtilis 1A221 through enhanced expression of lmrAB has been reported (7). Additionally, 19 spontaneous lincomycin-resistant mutants have been isolated and have also been shown to elevate the expression of lmrAB (11).…”

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Bacillus subtilisLmrA Is a Repressor of thelmrABandyxaGHOperons: Identification of Its Binding Site and Functional Analysis oflmrBandyxaGH

et al. 2004

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The Bacillus subtilis lmrAB operon is involved in multidrug resistance. LmrA is a repressor of its own operon, while LmrB acts as a multidrug efflux transporter. LmrA was produced in Escherichia coli cells and was shown to bind to the lmr promoter region, in which an LmrA-binding site was identified. Genome-wide screening involving DNA microarray analysis allowed us to conclude that LmrA also repressed yxaGH, which was not likely to contribute to the multidrug resistance. LmrA bound to a putative yxaGH promoter region, in which two tandem LmrA-binding sites were identified. The LmrA regulon was thus determined to comprise lmrAB and yxaGH. All three LmrA-binding sites contained an 18-bp consensus sequence, TAGACCRKTCWMTATAWT, which could play an important role in LmrA binding

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“…Media, transforming DNA, and competent cells were prepared as described (7). Selection for antibiotic resistance (8) and DNA-mediated transformation (7) were as described.…”

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confidence: 99%

Thiostrepton-resistant mutants exhibit relaxed synthesis of RNA.

Smith¹,

Paress²,

Pestka³

1978

Proc. Natl. Acad. Sci. U.S.A.

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Spontaneous mutants -of Bacillus subtifis resistant to thiostrepton (tsp) exhibit relaxed synthesis of RNA when starved for required amino acids. Intact cells of tsp mutants cannot synthesize the regulatory nucleotides, ppGpp and pppGpp, after amino acid deprivation. Because ribosomes isolated from spontaneous revertants to thiostrepton sensitivity and from wild-type stringent strains can synthesize (p)ppGpp whereas ribosomes isolated from tsp strains cannot synthesize these regulatory nucleotides in the presence of stringent factor, it appears that the lesion is expressed at the level of the ribosome. Genetic mapping, via three-factor transformational crosses, has shown that tsp is closely linked to rif, in the order cysA14, tsp, rif-1, strA. The phenotype of the tsp mutants indicates that they are of the relC type. Their map position indicates that they are different from a previously described B. subtilis rel mutation. Ribosomes from the latter strain can synthesize (p)ppGpp in cell-free extracts.Bacillus subtilis is normally sensitive to thiostrepton (also known as bryamycin), an antibiotic that inhibits several ribosomal functions, including initiation, elongation factor Tu-and Gdependent reactions, and termination (1). Spontaneous mutations to thiostrepton resistance (tsp) have been isolated in B. subtilis, and ribosomes isolated from tsp strains are resistant to the antibiotic (2). Resistance to thiostrepton is expressed at the level of the 50S subunit, as shown by GTP binding and protein synthesis in cell-free extracts (3, 4). Immunochemical analysis and two-dimensional gel electrophoresis have shown that the 50S protein BS LII of B. subtilis is immunologically related to Escherichia coli LIi and is missing from the ribosomes of tsp strains (unpublished data). Ribosomal protein Lii is altered in E. coli reiC mutants, which are unable to synthesize the regulatory nucleotides ppGpp and pppGpp when starved for amino acids (5, 6). In addition, ribosomes from relC strains do not synthesize ppGpp and pppGpp in extracts (6). Because tsp mutants seem to lack protein BS LIi, it was possible that tsp mutants, derived from stringent B. subtilis parental strains, would exhibit a relaxed phenotype in response to amino acid starvation and that ribosomes derived from tsp strains, like ribosomes isolated from reiC mutants, would be unable to synthesize ppGpp and pppGpp in extracts. This report examines these predictions.EXPERIMENTAL PROCEDURES Genetic Methods. Media, transforming DNA, and competent cells were prepared as described (7). Selection for antibiotic resistance (8) and DNA-mediated transformation (7) were as described.Labeling of Intact Cells. B. subtilis strains IS75 and IS127 (Table 1) were grown at 370C in low phosphate medium con-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 5993taining the following ingredients in g/liter unless othe...

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Genetic Mapping of Antibiotic Resistance in Markers Bacillus subtilis

Cited by 65 publications

References 16 publications

Loss of Ribosomal Protein L11 Blocks Stress Activation of the Bacillus subtilis Transcription Factor ς ^B

Loss of Ribosomal Protein L11 Blocks Stress Activation of the Bacillus subtilis Transcription Factor ς ^B

Bacillus subtilisLmrA Is a Repressor of thelmrABandyxaGHOperons: Identification of Its Binding Site and Functional Analysis oflmrBandyxaGH

Thiostrepton-resistant mutants exhibit relaxed synthesis of RNA.

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Genetic Mapping of Antibiotic Resistance in Markers Bacillus subtilis

Cited by 65 publications

References 16 publications

Loss of Ribosomal Protein L11 Blocks Stress Activation of the Bacillus subtilis Transcription Factor ς B

Loss of Ribosomal Protein L11 Blocks Stress Activation of the Bacillus subtilis Transcription Factor ς B

Bacillus subtilisLmrA Is a Repressor of thelmrABandyxaGHOperons: Identification of Its Binding Site and Functional Analysis oflmrBandyxaGH

Thiostrepton-resistant mutants exhibit relaxed synthesis of RNA.

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Loss of Ribosomal Protein L11 Blocks Stress Activation of the Bacillus subtilis Transcription Factor ς ^B

Loss of Ribosomal Protein L11 Blocks Stress Activation of the Bacillus subtilis Transcription Factor ς ^B