Activation of the Escherichia coli marA/soxS/rob regulon in response to transcriptional activator concentration

Abstract

The paralogous transcriptional activators MarA, SoxS, and Rob activate a common set of promoters, the marA/soxS/rob regulon of Escherichia coli, by binding a cognate site (marbox) upstream of each promoter. The extent of activation varies from one promoter to another and is only poorly correlated with the in vitro affinity of the activator for the specific marbox. Here, we examine the dependence of promoter activation on the level of activator in vivo by manipulating the steady-state concentrations of MarA and SoxS in Lon protease mutants and by measuring promoter activation using lacZ transcriptional fusions. We found that: (i) the MarA concentrations needed for half-maximal stimulation varied by at least 19-fold among the 10 promoters tested; (ii) most marboxes were not saturated when there were 24,000 molecules of MarA per cell; (iii) the correlation between the MarA concentration needed for half-maximal promoter activity in vivo and marbox binding affinity in vitro was poor; and (iv) the two activators differed in their promoter activation profiles. The marRAB and sodA promoters could both be saturated by MarA and SoxS in vivo. However, saturation by MarA resulted in greater marRAB and lesser sodA transcription than did saturation by SoxS, implying that the two activators interact with RNA polymerase in different ways at the different promoters. Thus, the concentration and nature of activator determine which regulon promoters are activated, as well as the extent of their activation.

Figure 1

MarA molecules per cell as a function of IPTG concentration in wild type and lon− clpP−cells. The inset is one of many Western blots from which the values in the graph were calculated. As previously noted, MarA made in vivo migrates slightly faster than the purified material from the plasmid construct which contains an additional 5 amino acids at its N-terminus. There is a band also present with pre-immune serum that moves slightly less rapidly than the MarA. Each lane contained the extract from 4.5*10⁷ lon− clp− cells (M3897) or 1.4*10⁸ wild-type cells (M3723). The two MarA lanes contained 11 (left lane) and 33 ng (right lane) of purified MarA.

Figure 2

The β-galactosidase activities of 10 promoters of the marA/soxS/rob regulon as a function of the calculated number of MarA molecules in lon− clpP− cells. The promoter::lacZ fusions are indicated.

Figure 3

Activation of three promoters by MarA or SoxS as a function of IPTG concentration in wild-type (A, C, E) and in lon− clpP− cells (B, D, F). marRAB promoter (A, B); sodA promoter (C, D); micF promoter (E, F). Circles, MarA; squares, SoxS; open symbols, wild-type cells; closed symbols, lon− clpP− cells.