Regulation of RpoS proteolysis in Escherichia coli: The response regulator RssB is a recognition factor that interacts with the turnover element in RpoS (Sigma-Sysigma factoryClp proteaseytwo-component systemystress)

The degradation of the RpoS (sS) 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 or sS 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-bgalactosidase 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 location of the turnover element in RpoS and its molecular function have not been demonstrated. To understand the recognition of RpoS as a substrate for the proteolytic machinery, we have localized the turnover element by site-directed mutagenesis and have used the resulting mutants to characterize the molecular function of this element both in vivo and in vitro. From the data presented here, we conclude that the turnover element in RpoS (with K173 as an essential amino acid) is a binding site for the response regulator RssB. Moreover, binding of RssB to RpoS is modulated by phosphorylation of the RssB receiver domain. MATERIALS AND METHODS Bacterial Strains, Plasmids, and Growth Conditions. The strains used in this study are derivatives of MC4100 (15), into which various alleles of rpoS, rssB, and clpP were introduced by P1 transduction (16). Specifically, these alleles are rpoS359::Tn10 (17), rssB::Tn10 (11), rssB::cat (kindly provided by F. Moreno, Hospital Ramón y Cajal, Madrid), and clpP1::cat (18). Strains carrying reporter gene fusions to RpoS-dependent genes were the following: RO151(MC4100 carrying csi-5(osmY)::lacZ(lplacMu55); ref. 19), RH95 (MC4100 carrying lMAV103:: bolAp1::lacZYA; ref. 20), DW12 (MC4100 carrying csi-12(csiD)::lacZ(lplacMu15); ref. 19), LB83 (MC4100 carrying otsB::lacZ(lplacMu55), with the otsB fusion derived from FF1112 (21), and their respective rpoS, rssB, and clpP derivatives obtained as mentioned above. These strains were used as recipients for plasmids derived from pBAD18, which express different variants of RpoS under the control of the pBAD promoter (see below). Cultures were grown at 37°C under aeration in LB medium or in minimal medium M9 (16) supplemented with 0.4% glycerol. Ampicilline (100 mg ml21) was used to grow plasmidcontaining strains. For selecting transductants, various antibiotics were added as recommended (16). Growth was monitored by measuring the OD at 578 nm. Introduction of Single Amino Acid Substitutions in RpoS by in Vitro Mutagenesis. For the isolation of rpoS mutations by site-directed mutagenesis, an EcoRI–HindIII fragment carrying the rpoS structural gene was obtained from pRL40.1 (which contained rpoS under the control of the ptac promoter; ref. 4) and cloned into the multiple cloning site of pBAD18 directly downstream of the pBAD promoter, which yielded pRpoS18. For replacement of a rpoS-internal fragment containing the putative turnover element, unique XmaIII and The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. PNAS is available online at www.pnas.org. *G.B. and E.K. contributed equally to this work. †To whom reprint requests should be addressed at: Institute of Plant Physiology and Microbiology, Free University of Berlin, KöniginLuise-Str. 12–16a, 14195 Berlin, Germany. e-mail: Rhenggea@ zedat.fu-berlin.de.