Presence of Guanosine 5'-Diphosphate 3'-Diphosphate in Bacillus subtilis Vegetative Cells

Abstract

Extracts of Bacillus subtilis vegetative cells with 2 M formic acid contained a large amount of a hyperphosphorylated nucleotide ("spot 4" nucleotide). The compound always comigrated with authentic guanosine 5'-diphosphate 3'-diphosphate (ppGpp) on two-dimensional polyethyleneimine (PEI)-cellulose thin-layer chromatography performed with three different solvent systems. Furthermore, all dephosphorylated 32P-labeled derivatives from the "spot 4" nucleotide comigrated on a one-dimensional PEI-cellulose plate with those from authentic ppGpp present in the same reaction mixture, when the compounds were hydrolyzed with snake venom phosphodiesterase or alkali. The level of the "spot 4" nucleotide (ppGpp) in the cell extracts was 0.14 nmol P/A660, corresponding to about one-third of the guanosine 5'-triphosphate (GTP) level and about 10% of the adenosine 5'-triphosphate (ATP) level. These results indicate that a "magic spot" nucleotide, ppGpp, is present at a high level in B. subtilis cells vegetatively growing in mNSMP.

Involvement of the stringent response in degradation of glutamine phosphoribosylpyrophosphate amidotransferase in Bacillus subtilis

“…We have shown that 3H-labeled pppGpp and ppGpp are stable under our extraction conditions. Other authors (9)(10)(11) have also suggested that extraction with 0.1 M formic acid does not extract (p)ppGpp quantitatively from bacterial cells. These authors have adopted procedures such as centrifugation of the cells and extraction by incubation with detergents or lysozyme.…”

“…Relation of our findings to observations of others who have studied (p)ppGpp levels in bacilli. A number of laboratories have reported measurements of intracellular (p)ppGpp levels in Bacillus species under many of the conditions that we have examined (1,9,10,(12)(13)(14)(15)20). However, these groups have not all used the same extraction procedures, culture media, or bacterial strains that we have used to study amidotransferase degradation.…”

Four Phosphates at One Blow: Access to Pentaphosphorylated Magic Spot Nucleotides and Their Analysis by Capillary Electrophoresis

“…14 In contrast to (p)ppGpp, the existence and role of adenosine MSN in bacteria are controversially discussed since the 1970s. 15,16 More recent reports from Bruhn-Olszewska et al demonstrated in vitro how pppApp (4) has opposite regulatory effects compared to ppGpp (1) when binding to E. coli RNA polymerase. Furthermore, pppApp (4) is a stronger effector than ppApp (2).…”

Identification of an Indispensable Amino Acid for ppGpp Synthesis ofEscherichia coliSpoT Protein

“…The results of direct detection of ppGpp synthesis [27][28][29][30] Nucleotides from the cells were extracted with 1 M formic acid, 31) and then were separated by two-dimensional PEI thin-layer chromatography (Polygram CEL 300 PEI from Macherey Nagel Co.). Solvents used for the chromatography were as follows:ˆrst dimension; 3.3 M ammonium formate, 0.68 M boric acid (pH 7.0), and second dimension; 1.5 M potassium phosphate (pH 3.4).…”

“…Solvents used for the chromatography were as follows:ˆrst dimension; 3.3 M ammonium formate, 0.68 M boric acid (pH 7.0), and second dimension; 1.5 M potassium phosphate (pH 3.4). [31][32][33] The plates were used to expose Fuji-RX X-rayˆlm for 3 days and theˆlm was developed. Arrows and arrowheads indicate the spots of ppGpp and pppGpp, respectively.…”