S produces a fine-tuned checkpoint system that "unlocks" curli expression only after down-regulation of flagellar gene expression. In summary, these data reveal the logic and sequence of molecular events underlying the motile-to-adhesive "lifestyle" switch in E. coli.[Keywords: Adhesion; c-di-GMP; flagella; GGDEF; EAL; RpoS] Supplemental material is available at http://www.genesdev.org.

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...

. However, when s S is ectopically expressed in exponential phase, it shows only basal activity (Kvint et al., 2000). Here, we try to highlight how Es S manages to activate its regulon in vivo only in physiologically appropriate conditions. In general, Es S efficiency depends on the cellular s S concentration (controlled at the levels of transcription, translation and proteolysis) and Es S holoenzyme formation and activity, whereas Es S promoter specificity is achieved by a combination of cis-and trans-acting promoter features. Intrinsic promoter features support distinct promoter utilization by Es S and Es 70

Switching from the motile planktonic bacterial lifestyle to a biofilm existence is stimulated by the signalling molecule bis-(3′-5′)-cyclic-diguanosine monophosphate (cyclic-di-GMP), which is antagonistically controlled by diguanylate cyclases (DGCs; characterized by GGDEF domains) and specific phosphodiesterases (PDEs; mostly featuring EAL domains). Here, we present the expression patterns of all 28 genes that encode GGDEF/EAL domain proteins in Escherichia coli K-12. Twenty-one genes are expressed in Luria–Bertani medium, with 15 being under σ S control. While a small subset of GGDEF/EAL proteins (YeaJ and YhjH) is dominant and modulates motility in post-exponentially growing cells, a diverse battery of GGDEF/EAL proteins is deployed during entry into stationary phase, especially in cells grown at reduced temperature (28 °C). This suggests that multiple signal input into cyclic-di-GMP control is particularly important in growth-restricted cells in an extra-host environment. Six GGDEF/EAL genes differentially control the expression of adhesive curli fimbriae. Besides the previously described ydaM, yciR, yegE and yhjH genes, these are yhdA (csrD), which stimulates the expression of the DGC YdaM and the major curli regulator CsgD, and yeaP, which contributes to expression of the curli structural operon csgBAC. Finally, we discuss why other GGDEF/EAL domain-encoding genes, despite being expressed, do not influence motility and/or curli formation.

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.