Fig. 5.

In vivo and in vitro effects of the synthesized peptides. (A) In vivo effects. The effects on PspoIIG promoter activity in DSM, when only rapH is overexpressed, of the indicated molar concentrations of synthesized TDRNTTY (diamonds), TDRNTT (triangles), DRNTTY (circles), and DRNTT (squares) are presented as a percentage of the wild-type activity. The wild-type reference is the level of PspoIIG promoter activity after 5.5 h of growth when both rapH and phrH are overexpressed. For the full curves of expression, see Fig. S4 in the supplemental material. (B) In vitro inhibition of RapH-mediated Spo0F dephosphorylation by synthesized peptides. 6 μM ³²P-labeled Spo0F∼P, 6.5 μM RapH, and PhrH at the indicated concentrations were incubated at 25°C. The autodephosphorylation of Spo0F, in the absence of RapH (•) (labeled Spo0F autodephosphorylation) was similarly measured at 25°C. Aliquots were removed from the reaction at the indicated time points and visualized by phosphorimaging. The concentrations of TDRNTT used were as follows: 0 μM (labeled no PhrH) (▪), 6.5 μM (▾), 65 μM (▴), and 650 μM (⧫). Phosphatase activity data were fit to exponential curves using KaleidaGraph software (Synergy Software). The dashed curve shows autodephosphorylation. Gels depicting Spo0F dephosphorylation in the absence of RapH, the presence of RapH, or the presence of RapH plus peptide are also presented. The additional gels used to construct this figure are shown in Fig. S5 in the supplemental material.