FIGURE 3.

Accumulation of intracellular ppGpp is specifically induced during anaerobic TMAO respiration, and CT production is regulated by the level of intracellular ppGpp. A, intracellular nucleotides were detected by using TLC analysis. Wild type N16961 was grown in LB, LBT, or LBF, with [³²P]orthophosphate for 8 h. The ΔrelA ΔspoT double mutant and ΔrelA ΔspoT ΔrelV triple mutant (ppGpp null mutant, termed ppGpp°) were grown in LBT with [³²P]orthophosphate for 8 h. Cellular extracts were prepared and analyzed in TLC. B, strains indicated at the left were grown in LB, LBT, and LBF, and aliquots were sampled for cfu counting. Experimental conditions were identical to those described in the legend to Fig. 2B. C, the levels of CT produced in N16961, ΔrelA ΔspoT, and ppGpp° mutants. Strains were grown in LBT for 16 h, and culture supernatants were assayed for CT ELISA. Experimental conditions were identical to those described in the legend to Fig. 1. D, the level of CT produced in N16961, ΔrelA ΔspoT, and ppGpp° mutants. Strains were grown in AKI conditions, as described under “Experimental Procedures,” and culture supernatants were assayed for CT ELISA. Error bars, S.D.