guanosine-triphosphate and serine-hydroxamate

Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of bacteria to amino acid starvation, and has thus been termed a bacterial alarmone. Previous X-ray crystallographic analysis has provided a structural basis for the transcriptional regulation of RNA polymerase activity by ppGpp in the thermophilic bacterium Thermus thermophilus. Here we investigated the physiological basis of the stringent response by comparing the changes in intracellular ppGpp levels and the rate of RNA synthesis in stringent (rel(+); wild type) and relaxed (relA and relC; mutant) strains of T. thermophilus. We found that in wild-type T. thermophilus, as in other bacteria, serine hydroxamate, an amino acid analogue that inhibits tRNA(Ser) aminoacylation, elicited a stringent response characterized in part by intracellular accumulation of ppGpp and that this response was completely blocked in a relA-null mutant and partially blocked in a relC mutant harboring a mutation in the ribosomal protein L11. Subsequent in vitro assays using ribosomes isolated from wild-type and relA and relC mutant strains confirmed that (p)ppGpp is synthesized by ribosomes and that mutation of RelA or L11 blocks that activity. This conclusion was further confirmed in vitro by demonstrating that thiostrepton or tetracycline inhibits (p)ppGpp synthesis. In an in vitro system, (p)ppGpp acted by inhibiting RNA polymerase-catalyzed 23S/5S rRNA gene transcription but at a concentration much higher than that of the observed intracellular ppGpp pool size. On the other hand, changes in the rRNA gene promoter activity tightly correlated with changes in the GTP but not ATP concentration. Also, (p)ppGpp exerted a potent inhibitory effect on IMP dehydrogenase activity. The present data thus complement the earlier structural analysis by providing physiological evidence that T. thermophilus does produce ppGpp in response to amino acid starvation in a ribosome-dependent (i.e., RelA-dependent) manner. However, it appears that in T. thermophilus, rRNA promoter activity is controlled directly by the GTP pool size, which is modulated by ppGpp via inhibition of IMP dehydrogenase activity. Thus, unlike the case of Escherichia coli, ppGpp may not inhibit T. thermophilus RNA polymerase activity directly in vivo, as recently proposed for Bacillus subtilis rRNA transcription (L. Krasny and R. L. Gourse, EMBO J. 23:4473-4483, 2004).

Topics: Adaptation, Physiological; Adenosine Triphosphate; Amino Acid Sequence; Anti-Bacterial Agents; Bacterial Proteins; DNA-Directed RNA Polymerases; Gene Expression Regulation, Bacterial; Guanosine Tetraphosphate; Guanosine Triphosphate; IMP Dehydrogenase; Ligases; Molecular Sequence Data; Mutation; Promoter Regions, Genetic; Ribosomal Proteins; Ribosomes; RNA, Bacterial; RNA, Ribosomal, 23S; RNA, Ribosomal, 5S; Sequence Alignment; Serine; Tetracycline; Thermus thermophilus; Thiostrepton; Transcription, Genetic; Uracil

The accumulation of RNA and protein and the kinetics of nucleoside triphosphate and guanosine polyphosphate pools during amino acid starvation and carbon source downshift were investigated in Streptomyces hygroscopicus. RNA accumulation was controlled stringently during both amino acid starvation and carbon source downshift. The pool size of ppGpp increased dramatically under these conditions. However, the intracellular concentrations of nucleoside triphosphates were low and the concentration of guanosine polyphosphates was much lower than in Escherichia coli. The possible significance of this phenomenon in the regulation is discussed.

Topics: Adenosine Triphosphate; Amino Acids; Autoradiography; Bacterial Proteins; Guanosine Pentaphosphate; Guanosine Tetraphosphate; Guanosine Triphosphate; Methylglucosides; Methylglycosides; Nucleotides; RNA, Bacterial; Serine; Streptomyces

Lack of three different amino acids or treatment with the analogue DL-serine hydroxamate does not induce the accumulation of ppGpp and pppGpp, the 3'-pyrophosphates of GDP and GTP, respectively, in Rhizobium meliloti strain 41. Surprisingly, RNA accumulation is controlled under the above mentioned conditions stringently. Moreover, no significant RNA accumulation was found during chloramphenicol, tetracycline, and streptomycin treatment, suggesting that R. meliloti, unlike any other bacteria in investigated so far, is not able to accumulate RNA without ongoing protein synthesis. On the other hand, lack of carbon source and ammonium starvation result in a significant ppGpp accumulation.

Topics: Adenosine Triphosphate; Amino Acids; Bacterial Proteins; Chloramphenicol; Guanine Nucleotides; Guanosine Tetraphosphate; Guanosine Triphosphate; Kinetics; Phenylalanine; Rhizobium; RNA, Bacterial; Serine