Extraction and detection of guanosine 5'-diphosphate-3'-diphosphate in amino acid starvation cells of Clavibacter michiganensis.

Guanosine 5'-diphosphate-3'-diphosphate (ppGpp) is a small molecule nucleotide alarmone that can accumulate under the amino acid starvation state and trigger the stringent response. This study reported the extraction of ppGpp from the Gram-positive bacteria Clavibacter michiganensis through methods using formic acid, lysozyme, or methanol. Following extraction, ppGpp was detected through ultra-high-performance liquid chromatography (UHPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The methanol method showed the highest extraction efficiency for ppGpp among the three methods tested. C. michiganensis cells in exponential growth phase was induced in amino acid starvation by serine hydroxamate (SHX) and used for ppGpp extraction and detection. When using the methanol extraction method, the results showed that ppGpp concentrations in SHX-treated samples were 15.645 nM, 17.656 nM, 20.372 nM, and 19.280 nM at 0 min, 15 min, 30 min and 1 h, respectively, when detected using LC-MS/MS. This is the first report on ppGpp extraction and detection in Clavibacter providing a new idea and approach for nucleotide detection and extraction in bacteria.

Response of RNA polymerase to ppGpp: requirement for the omega subunit and relief of this requirement by DksA.

Previous studies have come to conflicting conclusions about the requirement for the omega subunit of RNA polymerase in bacterial transcription regulation. We demonstrate here that purified RNAP lacking omega does not respond in vitro to the effector of the stringent response, ppGpp. DksA, a transcription factor that works in concert with ppGpp to regulate rRNA expression in vivo and in vitro, fully rescues the ppGpp-unresponsiveness of RNAP lacking omega, likely explaining why strains lacking omega display a stringent response in vivo. These results demonstrate that omega plays a role in RNAP function (in addition to its previously reported role in RNAP assembly) and highlight the importance of inclusion of omega in RNAP purification protocols. Furthermore, these results suggest that either one or both of two short segments in the beta' subunit that physically link omega to the ppGpp-binding region of the enzyme may play crucial roles in ppGpp and DksA function.

A unique class of compound, guanosine-nucleoside tetraphosphate G(5')pppp(5')N, synthesized during the in vitro transcription of cytoplasmic polyhedrosis virus of Bombyx mori. Structural determination and mechanism of formation.

Two structurally different classes of oligonucleotides accumulate in vitro in cytoplasmic polyhedrosis virus (CPV) transcription mixtures in molar excess as compared to the completed RNA products. The first class consists of oligonucleotides which correspond to the 5'-terminal sequence of the virus mRNAs (referred to as initiator oligonucleotides). The major species of initiator oligonucleotides are (p)ppApG and (p)ppApGpN together with smaller amounts of homologous capped structures (Furuichi, Y. (1981) J. Biol. Chem. 256, 483-493). In addition to initiator oligonucleotides, CPV transcription mixtures yielded a second new class of compounds which were radiolabeled by [alpha-32P]GTP and resistant to phosphatase digestion. Their structures were identified as G(5')pppp(5')A, G(5')pppp(5')C, G(5')pppp(5')G, and G(5')pppp(5')U. With the exception of G(5')pppp(5')G, these compounds have not been observed previously. The mechanism of synthesis of these unique compounds was elucidated as pppG + pppN leads to GppppN + PPi. The reaction resembles, in principle, a guanylylation reaction which occurs during cap formation in CPV and other eukaryotic mRNA syntheses. It is likely that these compounds are formed in a similar way by a condensation reaction involving a viral guanylyltransferase-pG intermediate complex and ribonucleoside triphosphate. When the amounts of G(5')pppp(5')N were measured, it was found that G(5')pppp(5')N reached maximum concentrations (0.4 to 0.7 microM) shortly after the onset of RNA synthesis (1 h) and these levels were maintained or diminished gradually. By contrast, mRNA and (p)ppApG were continuously synthesized. The relative molar ratios of total G(5')pppp(5')N and (p)ppApG versus mRNA were comparable (74:24:1 and 30:27:1 during 1 to 4 h transcription, respectively). The results imply that these unusual compounds G(5')pppp(5')N as well as initiator oligonucleotides may be produced reiteratively during initiation when RNA chain elongation and capping are uncoupled.