Thiostrepton-resistant mutants exhibit relaxed synthesis of RNA.

Abstract

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LII of B. subtilis is immunologically related to Escherichia coli LIi and is missing from the ribosomes of tsp strains (unpublished data).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...

Kirromycin‐resistant elongation factor Tu from wild‐type of Lactobacillus brevis

“…Mutants of Escherichia coli and Bacillus subtilis selected for resistance to kirromycin have been shown to possess an altered form of EF-Tu [3][4][5]. Acquisition of kirromycin resistance introduces functionally related modifications of the EF-Tu nucleotide-binding site [3,4].…”

Induction of cat-86 by chloramphenicol and amino acid starvation in relaxed mutants of Bacillus subtilis

“…The lack of cat-86 induction in the reiC mutant strain was probably not due to the relaxed phenotype of the cells, since the relA mutation did permit drug inductioh of cat-86. Rather, we suspect that the structural alteration of the ribosome in reiC mutants may prevent the ribosomal function that is involved in destabilization of the RNA stem-loop (1,18).…”

“…The relevant mutation is within a gene designated reiC, which maps to the ribosomal protein gene cluster (9). Mutations in reiC result in the absence of protein Lii from the large ribosomal subunit (18). It seems reasonable to believe that Lii is part of a ribosomal protein complex involved in the synthesis of ppGpp.…”

“…In B. subtilis mutations at any of three loci confer the relaxed, or Rel, phenotype (9). The relA gene is thought to be the structural gene for stringent factor, and the reiC gene is believed to encode a ribosomal protein whose function is essential to the synthesis of ppGpp (17,18). The relG locus is less well defined, but this gene appears to be essential for the stringent response induced by glucose starvation of B. subtilis (14).…”

“…2 (18). The relevant mutation is within a gene designated reiC, which maps to the ribosomal protein gene cluster (9).…”