(p)ppGpp extraction and TLC.

The (p)ppGpp levels were checked by TLC (polyethyleneimine [PEI] cellulose) of formic acid extracts from the various strains. For in vivo labeling, the protocol described earlier (31) was used. Briefly, the bacterial culture was grown until an OD₆₀₀ of 0.2. The cultures were supplemented with 100 μCi of [³²P]H₃PO₄ and grown until an OD₆₀₀ of 0.9 to 1. The cells were harvested under cold conditions, washed once, resuspended in 20 to 30 μl of 10 mM Tris-HCl (pH 8), and treated with 50 mg/ml lysozyme and 1% SDS for 30 min on ice, followed by addition of 13 M (final concentration) HCOOH and 4 or 5 cycles of freeze-thaw for lysis. The extract was quickly centrifuged at 13,000 rpm for 10 min at 4°C. Aliquots (5 to 10 μl with equal radioactive counts) were spotted on PEI cellulose plates, developed with the mobile phase consisting of 1.5 M KH₂PO₄ (pH 3.4), and exposed to phosphorimaging plates for visualization/quantification. To generate markers, an in vitro (p)ppGpp synthesis assay was done by taking purified RelA (Rel-Msm-NTD protein) (18). (p)ppGpp was synthesized by adding RelA (1 μg) in the presence of GDP/GTP (1 mM), ATP (1 mM), 1 μCi of [γ-³²P]ATP for 1 h at 37°C in 20 μl buffer (40 mM Tris-Cl, pH 7.8, 2 mM DTT, 10 mM MgCl₂, 100 mM NH₄Cl). The reaction was stopped by 2% (final concentration) HCOOH and centrifuged at 13,000 rpm for 10 min at 4°C in a table-top Kubota 3500 centrifuge. The supernatants were collected separately and then diluted 200- to 500-fold in 13 M (final concentration) HCOOH and spotted (10 μl) on the TLC.