Fis Regulates Transcriptional Induction of RpoS in Salmonella enterica

Bacteria maintain intricate signaling networks that sense the environment and adjust cellular physiology accordingly. In Salmonella enterica serovar Typhimurium and Escherichia coli, less favorable growth conditions (e.g., nutrient limitation, starvation, low temperature, or osmotic shock) initiate a general stress response by triggering the synthesis of the RNA polymerase sigma factor RpoS (σS). By directing RNA polymerase to promoters of specific genes involved in stress resistance, RpoS serves as the central regulator of the general protective response, also known as stationary phase (SP), and thus increases survival (28). The importance of RpoS to serovar Typhimurium pathogenesis is evident from a mouse model involving lethal infections where rpoS mutants are completely avirulent (22). The complexity of RpoS regulation is illustrated by the variety of mechanisms reported thus far in E. coli: transcription, translation, protein turnover, and protein activity (28). One of the best-characterized induction phenomena is regulation of RpoS translation at low temperature in rich medium (≤30°C). This stimulus increases transcription of a regulatory RNA, DsrA, which can pair with an upstream antisense element in the leader region of the rpoS transcript to relieve the antisense element's inhibition of rpoS translation (38). This process requires the Sm-like RNA-binding protein, Hfq, and results in activation of RpoS expression at a posttranscriptional step. Notably, hfq mutants show normal SP induction of RpoS in rich medium, both in E. coli (29) and in serovar Typhimurium (our unpublished data). Another regulatory pathway limiting RpoS abundance in growing cells is proteolytic degradation involving the ATP-dependent ClpXP protease and a response regulator called MviA (in serovar Typhimurium) or SprE/RssB in E. coli (52, 62). In this pathway MviA is activated by an unknown stimulus through phosphorylation on D58, which substantially increases its ability to bind to RpoS. The relevant kinase has not yet been found (16, 28). The binding event (dependent on K173 of RpoS) results in a sequestered nonfunctional RpoS molecule and thus can modulate RpoS activity in itself (8, 75). The MviA-RpoS complex also interacts with the ClpXP protease, which then actively degrades RpoS, recycling MviA (44, 52). RpoS elevates transcription of the response regulator during SP, thus constituting an autoregulatory loop in which the concentration of MviA is a limiting factor for the rate of RpoS degradation in vivo (53, 57). Perhaps the most striking induction of RpoS is observed during growth to SP in rich medium, where the level of induction exceeds 30-fold, based on the activity of RpoS-responsive reporters and rpoS-lac fusions (28; unpublished data). The transcriptional component of this induction ranges from 5- to 10-fold (28, 29). Expression levels are significantly lower in the absence of guanosine tetraphosphate during both growth and SP, but the actual induction ratio is nearly unchanged (29). The cyclic AMP (cAMP) receptor protein, Crp, is also thought to be involved in rpoS transcriptional control, and yet the effect of the mutants is modest and interpretation is difficult due to the growth deficiency of the crp or cya (defective in adenylate cyclase) mutants in combination with growth rate transcriptional control of rpoS (36, 37). In the present study we show that the Fis protein (factor for inversion stimulation) is involved in RpoS regulation during growth in rich medium. Fis is a DNA-binding and bending protein that was initially characterized for its stimulatory role in site-specific DNA recombination (31, 34). Fis has been implicated in many other processes such as stimulation of excision and integration of lambda (4, 5, 20, 67), DNA replication at oriC (24, 26, 58), transposition (70), invasion of HEp-2 cells (71), and transcriptional activation and repression of several genes, including hns, leuV, gyr, tyrT, proP, nuo, osmE, and rRNA operons (3, 12, 21, 33, 56, 68, 72, 74). We investigate here rpoS promoter activity and demonstrate that Fis mutants have elevated expression of rpoS that is specific to exponential phase. This pattern of regulation is in good agreement with the known variation in Fis abundance in different phases of growth: Fis is undetectable in SP but is present at over 40,000 dimers per cell upon dilution into fresh medium (2, 6, 50). Based on these results and the requirement for specific sequences upstream of the major rpoS promoter, we present an intuitive model for transcriptional regulation of rpoS in which Fis binds to and represses transcription from PrpoS.