We have shown here that the extent to which genes of the marA/soxS/rob regulon are activated is a function of MarA concentration. Under steady-state conditions, intermediate levels of MarA fully activate some members of the regulon (e.g., marRAB, sodA) without significant activation of other members of the regulon (e.g., acnA, pqi5, ). While we have not measured the number of molecules of SoxS per cell that were produced by IPTG-treatment, the data ( and unpublished) indicate that the response of the regulon to SoxS concentration is similar.
We call this phenomenon “commensurate regulon activation” because it enables E. coli
to mount a proportionate response of the marA/soxS/rob
regulon to a stress signal, activating the minimum number of genes necessary for overcoming a prolonged threat. When there is a low level of signal, a low level of activator is made and only a few genes are activated (e.g., micF
which decreases outer membrane permeability and sodA
which converts superoxides to peroxides). When the stress is greater, more activator is made and additional genes are activated (e.g., mdaB, zwf, fpr
). Only at the highest stress levels are the highest activator levels made and the full panoply of genes brought into play (e.g., acrAB, tolC, pqi5
). Commensurate activation therefore enables the level of threat to be matched to the cost of a response. For example, over-production of the AcrAB–TolC efflux pump, essential for the removal of multiple antibiotics and organic solvents, may also deplete the cell of energy and vital constituents. If overexpression of these genes were not costly to the cell, we would expect wild-type, unthreatened cells to have higher basal levels of MarA, SoxS and Rob and/or higher basal levels of transcription of the regulon promoters. We presume that the comparatively low levels of basal transcription for each promoter is optimal in the absence of threat. Indeed, it has been observed that overexpression of MarA and SoxS or activation of Rob can lead to severe growth inhibition.13,14
Commensurate activation is likely relevant to transcriptional regulation of systems other than the marA/soxS/rob
regulon. For example, it has previously been suggested that different promoters of the CRP modulon are activated at different levels of cAMP-CRP, leading to an observed hierarchy of response to cAMP concentration15,16
although parallel measurements of promoter activity and cAMP-CRP concentration were not measured in those studies. In a further example, the response of different recA-dependent promoters to the same signal is diverse; however, in this case the diversity cannot be completely explained by differences in the way RecA acts at promoters.17
Because of the connections between the mechanisms of the steady-state
phenomenon of commensurate activation and the dynamic
phenomenon of temporally ordered activation,18–20
we would also expect activation of the marA
regulon to exhibit temporal ordering in response to a slow rise in MarA. However, salicylate and paraquat induce a rapid rise in MarA and SoxS levels.13,21
Furthermore, because promoters of the regulon control expression of functionally diverse genes, the temporal ordering might not lead to the advantages in efficiency proposed for temporal ordering of functionally coherent regulons.18–20
We also note that the first promoter activated by either activator is marRAB
This has two consequences: 1) MarA autocatalytically increases its own synthesis, which is a rare feature among transcription factors in E. coli
Positive self-regulation is associated with multistability 24,25
but we have seen no evidence for multistability in activation of the marA
regulon. It will be important to determine the functional consequences of positive autoregulation of MarA. 2) The SoxS signal is also converted into a MarA signal, tying the two responses together. Nevertheless, because of promoter discrimination, overexpression of MarA leads to greater antibiotic resistance and less superoxide resistance than does overexpression of SoxS.9
Thus, commensurate activation enables the cell to bring many different defenses into play depending on the kind of signal, its amplitude and its duration, providing a flexible defense against different levels of threat. It is also likely that in a population of cells there will be substantial heterogeneity in terms of the extent of activation of any particular regulon member and this may provide further advantages for survival.
We can use the relation between promoter activation and MarA concentration () to back-calculate from the β-galactosidase activity of a cell to an equivalent concentration in MarA for any regulon promoter. Thus, our standard treatment of wild-type cells with 5 mM salicylate for 1 hr is the net equivalent of achieving a steady-state concentration of about 9,000 molecules of MarA per cell, far higher than the 750 molecules measured previously. This is likely due to a systematic error in the Western blot analyses of lon+ clpP+ strains.
We estimate that, at a minimum, there is a 19-fold variation in the amount of MarA needed for half-saturation of the different promoters. This variation is only poorly correlated with the binding affinity of MarA with these promoters in vitro (). For example, as previously reported, the marboxes of pqi-5 and acrAB bind very tightly to purified MarA but pqi-5 and acrAB require relatively high concentrations of MarA for activation. In contrast, MarA binds the sodA promoter very weakly but sodA is activated by low concentrations of MarA. This suggests that other factors present in vivo such as DNA supercoiling and/or global regulators (Fis, H-NS, etc.) may play important roles in determining promoter response to activator.
We have used the term “discrimination” for differences in activation of a single promoter by the paralogous activators and have shown a rough correlation of activation with the affinity (KD
) of a particular activator for a particular marbox
However, at the highest activator concentrations attained here, both MarA and, as we have argued above, SoxS saturate the marRAB
and sodA marboxes
() so KD
is not a limiting factor. Nevertheless, the marRAB
promoter shows greater activity with MarA than with SoxS whereas the opposite is true for the sodA
promoter. One possibility is that the two activators differ in how they interact with RNAP at different promoters and that the specific ternary complex is critical.