The LysR Homolog LrhA Promotes RpoS Degradation by Modulating Activity of the Response Regulator SprE

Physiological adaptation to changing environmental conditions is essential to microbial viability. This is particularly important for bacteria that alternatively inhabit distinct niches differing in conditions of oxidative stress, temperature, pH, and osmolarity, as well as nutrient availability and exposure to various toxins. OmpF and OmpC, the major porins of the gram-negative bacterium Escherichia coli, allow nonspecific diffusion of solutes through the protective outer membrane. Because the abundant OmpF porin allows for the more rapid diffusion of solutes through the outer membrane, it is a major entry point for most molecules that E. coli encounters, contributing significantly to outer membrane permeability and thus susceptibility to environmental hazards (33). For this reason, regulation of OmpF synthesis is necessarily complex and responsive to nutrient limitation (27) and a variety of environmental conditions (34), the best understood being medium osmolarity (10, 11, 28, 35). Since OmpF synthesis is highly responsive to fluctuation in various environmental parameters, studying its regulation provides us with an opportunity to understand the sensing mechanisms and complex regulatory networks necessary for E. coli adaptive physiology. Synthesis of OmpF is responsive to the bacterial growth rate such that during rapid logarithmic growth OmpF levels are high, but as bacterial growth begins to be limited by nutrient availability, OmpF synthesis decreases (27). This, and many other stationary-phase-dependent alterations in envelope physiology, presumably aids in survival under conditions of decreased metabolic capacity by encapsulating the bacterium within a protective, less permeable cell envelope (19). In addition to protective envelope adaptations, synthesis of a core set of cytoplasmic proteins is induced, independently of the nature of the limiting nutrient, as E. coli enters into stationary phase, resulting in a multiple-stress-resistant physiological state. For instance, stationary-phase E. coli is resistant to heat and osmotic and oxidative shock, as well as treatment with alkylating agents, ethanol, and acidic or basic pH (24, 27). These protective functions (22, 27), and in particular the stationary-phase-dependent decrease in OmpF synthesis (27, 36), were found to be mediated by the alternative primary sigma factor ςS (ς38), encoded by the rpoS gene. RpoS-dependent stationary-phase regulation of OmpF synthesis is mediated at the transcriptional level, as indicated by the approximately three- to fivefold derepression of ompF′-lacZ expression observed in an rpoS null mutant specifically during stationary-phase growth (36). However, the precise mechanism by which RpoS promotes transcriptional repression at ompF is unclear. RpoS functions as a global regulator of many genes required for the physiological transition into stationary-phase growth, and as such, its levels are highly responsive to the bacterial growth rate. During rapid growth, RpoS is maintained at a low level. However, during the transition to stationary phase, RpoS levels rise dramatically. This strict growth phase regulation of RpoS accumulation is quite complex in that it has been proposed to be mediated at all levels of synthesis and stability and via multiple inputs (15). Expression of various rpoS′-lacZ operon fusions increases approximately four- to fivefold during entry into stationary phase in rich medium and is weakly induced in response to starvation for carbon, nitrogen, or phosphate in minimal medium (22). The weak starvation induction ratio seen with rpoS′-lacZ transcription fusions is in stark contrast to the strongly increased amount of RpoS observed under the same medium conditions (20), implying that there is significant posttranscriptional growth phase regulation of RpoS. In fact, a greater stationary-phase induction ratio of RpoS at the level of translation was demonstrated with various rpoS′-′lacZ protein fusions (20, 23, 26). However, it has recently been argued that the stationary-phase-dependent induction of RpoS occurs exclusively through regulation of its susceptibility to proteolysis (46). Logarithmic-phase RpoS accumulation is minimal due to rapid degradation by the ClpXP protease, which requires amino acid residues 173 to 188 of RpoS for target recognition (40). ClpXP-mediated degradation of RpoS absolutely requires the two-component response regulator SprE (36), also called RssB (29) or, in Salmonella typhimurium, MviA (1). Constitutively active alleles of sprE prevent full induction of RpoS as bacteria enter stationary phase (36), while sprE null mutants allow logarithmic-phase RpoS accumulation to levels nearly equivalent to those observed in stationary phase (29, 36). SprE functions upstream of ClpXP to promote RpoS degradation in a substrate-specific manner which leaves ClpXP protease activity unaltered (36). Thus, SprE is the first response regulator to be implicated in regulating protein degradation, and its novel C-terminal domain reflects this unique function. For all known two-component response regulators, activity is modulated through phosphorylation at a conserved aspartic acid residue (16, 43). The N-terminal receiver domain of SprE contains this conserved residue, and so SprE activity is also predicted to be modulated via phosphorylation. The phosphorylation of SprE at the conserved aspartic acid has been demonstrated in vitro with acetyl phosphate (5). Additionally, it has been shown that Δ(pta ackA) mutants, which can no longer synthesize acetyl phosphate, affect the in vivo function of SprE such that it no longer efficiently promotes RpoS degradation (5). This implies that it is the phosphorylated form of SprE that is functionally active. In the absence of acetyl phosphate, however, RpoS accumulation remains growth phase regulated, indicating that there must be an additional factor(s) that influences SprE activity. The additional growth phase signal(s) and effector molecule(s) that regulate SprE remain to be discovered. Here, we describe the identification and initial characterization of another component of the SprE/ClpXP pathway that functions to modulate SprE activity.