C-di-GMP—which is produced by diguanylate cyclases (DGC) and degraded by specific phosphodiesterases (PDEs)—is a ubiquitous second messenger in bacterial biofilm formation. In Escherichia coli, several DGCs (YegE, YdaM) and PDEs (YhjH, YciR) and the MerR-like transcription factor MlrA regulate the transcription of csgD, which encodes a biofilm regulator essential for producing amyloid curli fibres of the biofilm matrix. Here, we demonstrate that this system operates as a signalling cascade, in which c-di-GMP controlled by the DGC/PDE pair YegE/YhjH (module I) regulates the activity of the YdaM/YciR pair (module II). Via multiple direct interactions, the two module II proteins form a signalling complex with MlrA. YciR acts as a connector between modules I and II and functions as a trigger enzyme: its direct inhibition of the DGC YdaM is relieved when it binds and degrades c-di-GMP generated by module I. As a consequence, YdaM then generates c-di-GMP and—by direct and specific interaction—activates MlrA to stimulate csgD transcription. Trigger enzymes may represent a general principle in local c-di-GMP signalling.

The degradation of the RpoS ( S ) subunit of RNA polymerase in Escherichia coli is a prime example of regulated proteolysis in prokaryotes. RpoS turnover depends on ClpXP protease, the response regulator RssB, and a hitherto uncharacterized ''turnover element'' within RpoS itself. Here we localize the turnover element to a small element (around the crucial amino acid lysine-173) directly downstream of the promoter-recognizing region 2.4 in RpoS. Its sequence as well as its location identify the turnover element as a unique proteolysis-promoting motif. This element is shown to be a site of interaction with RssB. Thus, RssB is functionally unique among response regulators as a direct recognition factor in ClpXP-dependent RpoS proteolysis. Binding of RssB to RpoS is stimulated by phosphorylation of the RssB receiver domain, suggesting that environmental stress affects RpoS proteolysis by modulating RssB affinity for RpoS. Initial evidence indicates that lysine-173 in RpoS, besides being essential of RpoS proteolysis, may play a role in promoter recognition. Thus the same region in RpoS is crucial for proteolysis as well as for activity as a transcription factor. RpoS orS is a sigma subunit of RNA polymerase that is present at very low levels in exponentially growing Escherichia coli cells. In response to various stress conditions, RpoS is strongly up-regulated and activates 50-100 genes, which results in multiple stress resistance and other physiological and morphological alterations (for recent reviews, see refs. 1 and 2). The control of the cellular RpoS content occurs at the levels of rpoS transcription and translation as well as RpoS proteolysis. In exponentially growing cells, RpoS is a very unstable protein (with a half-life of approximately 2 min), but RpoS is stabilized in response to carbon starvation or shift to high osmolarity, high temperature, or low pH (3-7).Some trans-acting factors involved in the control of RpoS proteolysis have been described. The relevant protease is ClpXP (8), a complex ATP-dependent protease consisting of proteolytic (ClpP) and chaperone (ClpX) subunits that form a proteasome-like assembly (9, 10). In addition, a twocomponent-type response regulator, RssB (also termed SprE or MviA), is essential for RpoS degradation (3,11,12). The C-terminal output domain of RssB is unlike that of any other response regulator and also does not show similarity to other proteins of known function. So far, its molecular function has remained unknown. RpoS degradation in vivo is positively modulated by acetyl phosphate, which readily phosphorylates the D58 residue in the RssB receiver domain in vitro (13).In addition to these trans-acting factors, a ''turnover element'' within RpoS is required for its proteolysis. The turnover element confers instability upon other proteins, e.g., RpoS-␤-galactosidase hybrid proteins (5,8). Thus, it may be functionally comparable to proteolysis-promoting elements in various eukaryotic proteins, such as the ''destruction box '' or D-box (14). The exact loca...

No abstract

SummaryIn growing Escherichia coli cells, the master regulator of the general stress response, s S (RpoS), is subject to rapid proteolysis.

s S (RpoS), the master regulator of the general stress response in Escherichia coli, is a model system for regulated proteolysis in bacteria. s S turnover requires ClpXP and the response regulator RssB, whose phosphorylated form exhibits high af®nity for s S . Here, we demonstrate that recognition by the RssB/ClpXP system involves two distinct regions in s S . Region 2.5 of s S (a long a-helix) is suf®cient for binding of phosphorylated RssB. However, this interaction alone is not suf®cient to trigger proteolysis. A second region located in the N-terminal part of s S , which is exposed only upon RssB±s S interaction, serves as a binding site for the ClpX chaperone. Binding of the ClpX hexameric ring to s S -derived reporter proteins carrying the ClpX-binding site (but not the RssB-binding site) is also not suf®cient to commit the protein to degradation. Our data indicate that RssB plays a second role in the initiation of s S proteolysis that goes beyond targeting of s S to ClpX, and suggest a model for the sequence of events in the initiation of s S proteolysis.