Figure 4

Effect of various mutants and addition of 8-Br-cAMP, 8-Br-cGMP and thapsigargin on AdcA phosphorylation. A. AdcA is not phosphorylated in response to 8-Br-cAMP. Vegetative or 4 h-starved cells in KK2 buffer (16.5 mM KH₂PO₄, 3.8 mM K₂HPO₄, pH 6.2) were treated with 20 mM 8-Br-cAMP or 200 mM sorbitol. B. AdcA is not dependent on DokA. dokA null cells kept in nutritive medium or starved for 4 h in KK2 buffer were treated with 200 mM sorbitol. C. AdcA is partly phosphorylated in response to a combination of 8-Br-cGMP and thapsigargin. Vegetative KAx-3 cells were treated with 20 mM 8-Br-cGMP, 10 μM thapsigargin or a mix of 8-Br-cGMP/thapsigargin. D. The response of AdcA is not affected in sgc/gca double null mutant. The sgc/gca null strain disrupted for the 2 guanylate cyclases sGC and GCA were subjected to 200 mM sorbitol. In all tested conditions, the response of AdcA was followed by Western blot using anti-AdcA antibodies (A, B, C and D). The phosphorylation of STATc in the same conditions was detected using the 3H7 and CP22 antibodies and was used as a positive control of cell responsiveness (A and C).