Anti-adaptors provide multiple modes for regulation of the RssB adaptor protein

Abstract

RpoS, an RNA polymerase s factor, controls the response of Escherichia coli and related bacteria to multiple stress responses. During nonstress conditions, RpoS is rapidly degraded by ClpXP, mediated by the adaptor protein RssB, a member of the response regulator family. In response to stress, RpoS degradation ceases. Small anti-adaptor proteins-IraP, IraM, and IraD, each made under a different stress condition-block RpoS degradation. RssB mutants resistant to either IraP or IraM were isolated and analyzed in vivo and in vitro. Each of the anti-adaptors is unique in its interaction with RssB and sensitivity to RssB mutants. One class of mutants defined an RssB N-terminal region close to the phosphorylation site and critical for interaction with IraP but unnecessary for IraM and IraD function. A second class, in the RssB C-terminal PP2C-like domain, led to activation of RssB function. These mutants allowed the response regulator to act in the absence of phosphorylation but did not abolish interaction with anti-adaptors. This class of mutants is broadly resistant to the anti-adaptors and bears similarity to constitutively activated mutants found in a very different PP2C protein. The mutants provide insight into how the anti-adaptors perturb RssB response regulator function and activation.

Stress sigma factor RpoS degradation and translation are sensitive to the state of central metabolism

“…Chloramphenicol (200 μg/mL) or tetracylcline (100 μg/mL) was added. Samples were analyzed as described by Battesti et al (19). Values presented are the mean of at least three independent assays.…”

“…Anti-adaptors act by interacting with the limiting levels of RssB and preventing the interaction of RssB with RpoS (19). Therefore, a stabilization mechanism dependent on anti-adaptor titration of RssB can be bypassed by increasing the levels of RssB.…”

“…RpoS degradation by the ClpXP protease depends on the adaptor protein RssB. The major mechanism that leads to RpoS stabilization under different stress conditions is inactivation of RssB adaptor activity by anti-adaptor proteins IraP, IraD, and IraM (2,18,19). Thus, we investigated if the known anti-adaptors play a role in the stabilization of RpoS in the aceE::kan background.…”

“…Mutating the D58 residue of RssB to A or P decreases but does not eliminate RpoS degradation in vivo and is still subject to stabilization by the anti-adaptors (2,(18)(19)(20). The small-molecule acetyl phosphate can phosphorylate RssB and stimulate degradation in vivo and in vitro (19,20).…”

“…Mutating the D58 residue of RssB to A or P decreases but does not eliminate RpoS degradation in vivo and is still subject to stabilization by the anti-adaptors (2,(18)(19)(20). The small-molecule acetyl phosphate can phosphorylate RssB and stimulate degradation in vivo and in vitro (19,20). Acetyl phosphate is synthesized from acetyl CoA, and one major pathway for acetyl CoA synthesis is dependent and ΔaceE (BA334) were grown to an OD 600 of ∼0.3, protein synthesis was inhibited by chloramphenicol (Cm) addition, and RpoS levels were analyzed by Western blotting using an anti-RpoS antiserum.…”