Article Figures & Data
Figures
- FIG. 1.
Effect of serine hydroxamate on growth and ppGpp accumulation. T. thermophilus HB8 (grown in MTM medium at 70°C), B. stearothermophilus JCM 2501 (grown in NB medium at 50°C), and B. subtilis 168 (grown in NB medium at 37°C) were treated with serine hydroxamate (SH) (final concentration, 10 mM). Changes in intracellular ppGpp levels were measured using HPLC as described by Ochi (38). Arrows indicate the addition of serine hydroxamate. Open circles represent growth in the presence of serine hydroxamate.
- FIG. 2.
Comparison of the amino acid sequences of the ribosomal protein L11 (an rplK gene product) in bacteria. Arrowheads show the mutations conferring the RelC and/or Tspr phenotype in Thermus thermophilus (this study), Escherichia coli (67), Halobacterium halobium (48), and Streptomyces coelicolor A3(2) (64). Asterisks indicate identical amino acids.
- FIG. 3.
Changes in intracellular ppGpp, ATP, and GTP levels in the wild-type strain (HB8) and the relC and relA mutant strains after serine hydroxamate treatment. (A) Strains were grown to an OD650 of 0.5 (mid-exponential phase) in MTM medium, after which serine hydroxamate was added to a final concentration of 10 mM. Concentrations of ppGpp, ATP, and GTP were determined by HPLC analysis. Circles, wild-type strain HB8; triangles, relC mutant KO-546; squares, relA disruptant KO-571. (B) Strains were grown in chemically defined medium (medium 162) containing phenylalanine (as required) and isoleucine, valine, and leucine (to promote the growth of the relA disruptant). When the OD650 reached 0.5 (mid-exponential phase), cells were filtered with a membrane filter, transferred to the same chemically defined medium but lacking phenylalanine, and incubated for an additional 18 min. Nucleotide concentrations were determined as in panel A. Error bars indicate standard deviation. Circles, wild-type strain KO-572 (Phe−); squares, relA disruptant KO-652 (Phe− relA).
- FIG. 4.
RNA synthesis by wild-type (strain HB8) or relC or relA mutant T. thermophilus. Cells were grown to an OD650 of 0.5 (mid-exponential phase) in synthetic medium, after which [2-14C]uracil (0.1 μCi/ml, 100 μM) was added to the culture, with (open circles) or without (closed circles) serine hydroxamate (10 mM), and the incubation was continued for an additional 10 min with shaking.
- FIG. 5.
Determination of the levels of 23Sa-23Sb transcripts by RT-qPCR analysis following serine hydroxamate treatment. Culture conditions of strains are the same as in Fig. 3A, and 0.5 ng (for 23Sa-23Sb) or 50 ng (for rpoD) of total RNA was used for the reaction. Error bars indicate standard deviation from four experiments. Circles, wild-type strain HB8; squares, relA disruptant KO-571.
- FIG. 6.
Ribosome-dependent (p)ppGpp synthesis with in vitro assay system. (A) Synthesis of (p)ppGpp by purified ribosomes from T. thermophilus HB8 (and E. coli W3110 as a reference strain). The reaction was carried out for 45 min at various temperatures as described in Materials and Methods. (B) Comparison of in vitro (p)ppGpp synthesis between ribosomes from T. thermophilus wild-type (HB8), relC mutant (KO-564), and relA disruptant (KO-571) strains. Reactions were done at 50°C as for panel A. (C) Two-dimensional TLC analysis of nucleotides. Three microliters of reactions in panel B was subjected to analysis by the two-dimensional TLC method as described previously (25). (D) Effect of various antibiotics on in vitro pppGpp synthesis. Rifampin (Rif), tetracycline (Tet), or thiostrepton (Tsr) was added to the reaction mixture to a final concentration of 10 μg/ml, after which the reaction was run at 50°C for 30 min. As a negative control, dimethyl sulfoxide (DMSO), the solvent for the antibiotic solution, was also tested at a concentration of 4% (vol/vol). Intensities of the pppGpp spots were quantitated with Image Gauge version 3.41 software (Fuji Firm) and are indicated at the bottom.
- FIG. 7.
SDS-polyacrylamide gel electrophoresis of the RNA polymerase fraction at each purification step. Lane 1, fraction eluted from the pellet after polyethylenimine P-70 precipitation; lane 2, peak fraction after calf thymus DNA-cellulose affinity chromatography; lane 3, peak fraction after superose 6 HR 10/30 gel filtration. α, β, and β′ represent subunits of RNA polymerase; σ represents σ-factor.
- FIG. 8.
Effect of ppGpp, pppGpp, and ppApp on in vitro transcription from the rRNA promoter. Effect of the indicated nucleotides on transcription of the 23S/5S rRNA operon (23Sb rRNA operon) was examined using an in vitro transcription system with purified T. thermophilus HB8 RNA polymerase (see Materials and Methods). (A) Effect of ppGpp, pppGpp, and ppApp on in vitro transcription, determined by measuring the radioactivity of the membrane filter as described in Materials and Methods. As a negative control, the reaction was also run without a template (dotted line). (B) Gel electrophoretic separation of runoff transcripts from in vitro transcription reactions with the 23S/5S rRNA operon promoter. ppGpp was added at the final concentration of 0, 0.05, 0.1, 0.2, 0.5 or 1 mM, and the products were separated on denaturing polyacrylamide gel as described in Materials and Methods. Runoff and paused products are indicated. (C) Quantitative evaluation of the band intensities of runoff transcripts shown in panel B.
- FIG. 9.
Effects of GTP and ATP concentration on 23S/5S rRNA (23Sb gene) promoter activity in vitro. (A) In vitro transcription was carried out with 0.5 mM (each) ATP, GTP, CTP, and UTP, except when ATP or GTP was varied at the indicated concentrations. The transcripts were separated on denaturing polyacrylamide gel as for Fig. 8B. (B) Intensities of the runoff transcripts shown in panel A.
