guanosine-tetraphosphate and Starvation

In nutrient-starved bacteria, RelA and SpoT proteins have key roles in reducing cell growth and overcoming stresses. Here we identify functional SpoT orthologs in metazoa (named Mesh1, encoded by HDDC3 in human and Q9VAM9 in Drosophila melanogaster) and reveal their structures and functions. Like the bacterial enzyme, Mesh1 proteins contain an active site for ppGpp hydrolysis and a conserved His-Asp-box motif for Mn(2+) binding. Consistent with these structural data, Mesh1 efficiently catalyzes hydrolysis of guanosine 3',5'-diphosphate (ppGpp) both in vitro and in vivo. Mesh1 also suppresses SpoT-deficient lethality and RelA-induced delayed cell growth in bacteria. Notably, deletion of Mesh1 (Q9VAM9) in Drosophila induces retarded body growth and impaired starvation resistance. Microarray analyses reveal that the amino acid-starved Mesh1 null mutant has highly downregulated DNA and protein synthesis-related genes and upregulated stress-responsible genes. These data suggest that metazoan SpoT orthologs have an evolutionarily conserved function in starvation responses.

Topics: Amino Acid Motifs; Amino Acid Sequence; Amino Acids; Animals; Binding Sites; Catalytic Domain; Conserved Sequence; Drosophila melanogaster; Drosophila Proteins; Gene Deletion; Gene Expression Regulation; Genetic Complementation Test; Guanosine Tetraphosphate; Heat-Shock Proteins; Humans; Hydrolysis; Ligases; Manganese; Models, Molecular; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis; Protein Conformation; Pyrophosphatases; Starvation; Structure-Activity Relationship

The recently identified role of LeuO in the regulation of transcription has prompted us to search for the specific function(s) of LeuO in bacterial physiology. The cryptic nature of expression of leuO has previously limited such analysis. A conditional leuO expression was found when bacteria enter stationary phase and was shown to be guanosine 3',5'-bispyrophosphate-dependent. Multiple physiological events, including the stringent response, are induced upon the increase of the bacterial stress signal, guanosine 3',5'-bispyrophosphate. In this study, we tested whether LeuO was directly involved in the bacterial stringent response. LeuO was shown to be indispensable for growth resumption following a 2-h growth arrest caused by starvation for branched-chain amino acids in an E. coli K-12 relA1 strain. This result supports a functional role for LeuO in the bacterial stringent response.

Topics: Amino Acids, Branched-Chain; Blotting, Western; Cell Division; Codon; Escherichia coli; Escherichia coli Proteins; Guanosine Tetraphosphate; Molecular Sequence Data; Mutation; Phenotype; Plasmids; Promoter Regions, Genetic; Starvation; Time Factors; Transcription Factors

In this study, the effects of high levels of guanosine tetraphosphate (ppGpp) on the decay and RNA chain elongation kinetics of the bacteriophage lambda late transcript in Escherichia coli were examined in the absence of amino acid starvation. The accumulation, mRNA decay kinetics, and RNA chain elongation rate of the lambda late mRNA were determined after heat induction of lambdacI857 lysogens in the presence of high levels of ppGpp induced from a RelAalpha fragment-overproducing plasmid. The accumulation kinetics and elongation rate determinations of the late mRNA were made at long times after induction to allow a new steady state of transcriptional activities under conditions of elevated intracellular levels of ppGpp. The results indicate no prolonged or significant effect on either mRNA decay or the RNA chain elongation rate of the late mRNA as a result of elevated ppGpp levels. Surprisingly, the RNA chain elongation rate determinations indicate an RNA polymerase processivity of approximately 90-100 nucleotides/s for the lambda late transcript despite the presence of high levels of ppGpp. The results are discussed in terms of various models for regulation of stable and messenger RNA synthesis in E. coli.

Topics: Amino Acids; Bacteriophage lambda; Escherichia coli; Guanosine Tetraphosphate; Hot Temperature; Isopropyl Thiogalactoside; Kinetics; Ligases; Lysogeny; Models, Genetic; Proviruses; RNA, Messenger; RNA, Viral; Starvation; Transcription, Genetic; Virus Activation

The response of marine Vibrio sp. strain S14 (CCUG 15956) to long-term (48-h) multiple-nutrient starvation (i.e., starvation for glucose, amino acids, ammonium, and phosphate simultaneously) can be described as a three-phase process. The first phase, defined as the stringent control phase, encompasses an accumulation of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and decreases in RNA and protein synthesis during the first 40 min. In the second phase, there is a temporary increase in the rates of RNA and protein synthesis between 1 and 3 h paralleling a decrease in the ppGpp pool. The third phase includes gradual decline in macromolecular synthesis after 3 h. Using two-dimensional gel electrophoresis of pulse-labeled proteins, a total of 66 proteins were identified as starvation inducible (Sti), temporally expressed throughout the three phases of starvation. The inhibition of protein synthesis during the first phase of starvation partly disrupted the subsequent temporally ordered synthesis of starvation proteins and prevented the expression of some late starvation proteins. It was also found that the early temporal class of starvation proteins, which included the majority of the Sti proteins, was the most essential for long-term survival. Vibrio sp. strain S14 cultures prestarved (1 h) for glucose, amino acids, ammonium, or phosphate as well as cultures exposed (1 h) to CdCl2 exhibited enhanced survival during the subsequent multiple-nutrient starvation in the presence of chloramphenicol or rifampin, while heat or the addition of cyclic AMP or nalidixic acid prior to starvation had no effect. It was demonstrated that amino acid starvation and CdCl2 exposure, which induced the stringent response, were the most effective in conferring enhanced survival. A few Sti proteins were common to all starvation conditions. In addition, the total number of proteins induced by multiple-nutrient starvation significantly exceeded the sum of those induced by starvation for each of the individual nutrients.

Topics: Bacterial Proteins; Chloramphenicol; Electrophoresis, Gel, Two-Dimensional; Energy Metabolism; Guanosine Tetraphosphate; Isoelectric Point; Marine Biology; Molecular Weight; Rifampin; RNA, Bacterial; Starvation; Vibrio