Detailed examination of the crystal structure of MarA suggested that the two regions of the molecule containing the two helix-turn-helix motifs were likely to be less flexible than those of SoxS (see Fig. S1 in the supplemental material). We therefore considered the possibility that the existence of bridging contacts formed between these two regions by E25 and R85 but absent in SoxS might account in part for the differences between MarA and SoxS with regard to the activation of different promoters. Thirteen MarA mutants (seven single-alanine substitutions, four charge inversion mutations [acidic to basic side chains or vice versa
] and two double-charge inversions) were compared with MarA and SoxS for activation of 16 MarA-SoxS-Rob regulon promoters. We reasoned that class I promoters were more likely to reveal a correlation between activity and flexibility, if it existed, since they depend on only a single interaction of MarA with the marbox and a single interaction with RNAP, whereas class II promoters involve additional interactions with RNAP. (The amino acids chosen for this study were known not to involve the interaction of MarA with RNAP [8
Only the E89A variant showed a consistent increase in activation of class I promoters that paralleled the activation by SoxS (Table ). This was surprising since E89A is unlikely to have any direct effect on the flexibility of MarA and was intended only as a control. As expected, there was no correlation between the activation of class II promoters by MarA(E89A) and by SoxS (Fig. ).
Activation of class I promoters by MarA, 13 MarA mutants, and SoxSa
To ascertain whether the greater activation of the acnA
, and zwf
promoters by MarA(E89A) was related to binding, SoxS, MarA, MarA(E89A), and MarA(Q91A) proteins were purified and assayed by gel retardation for their ability to bind the 20 bp marboxes (listed in Table ). In general, marbox binding (Fig. ) paralleled promoter activation for the class I promoters (Table ). Both SoxS and MarA(E89A) showed considerable binding to the marboxes of all of these promoters, whereas MarA and MarA(Q91A) showed significant binding to only the marRAB
marboxes. MarA(E89A) showed large increases in binding (relative to WT MarA), concomitant with large increases in relative activation for the acnA
, and zwf
promoters, and showed modest increases in both binding and activation of the acrAB
promoter. All four activators bound the marRAB
marbox with similar affinities and activated marRAB
to similar extents. SoxS bound the acrAB
marbox with similar affinity to MarA but, like MarA(E89A), marginally increased acrAB
transcription (~1.8-fold). Binding of the poxB
marbox was seen only for MarA(E89A) (data not shown). We note that, of these promoters, acnA
, and zwf
are necessary for optimal superoxide resistance (1
FIG. 2. Autoradiographs of gel electrophoretic mobility assays using MarA, MarA mutants E89A and Q91A, and SoxS with 32P-labeled 20-bp DNA fragments (Table ) corresponding to the marbox binding sites at different class I promoters. Partially (more ...)
Relative activation compared with relative binding for activators and mutants at MarA-SoxS-Rob regulon promotersa
The binding of MarA, the two MarA mutants, and SoxS to the marbox sequences of nine class II promoters was also determined by gel mobility assays and is summarized in Table . In every case, MarA(E89A) bound as well as or more tightly (i.e., had a lower KD
[equilibrium dissociation constant) than WT MarA. For several promoter marboxes this change was dramatic: the KD
150 to 50 for inaA
, from 200 to 75 for nfsB
, and from
150 nM to ~100 nM for both ybjC
. In spite of this, MarA(E89A) activation was greater than that of WT MarA for inaA
, and ybjC
; it was comparable to that of WT MarA for micF
and less than that of WT MarA for fumC
, and ybhW
(Table nd Fig. ). We conclude that the WT glutamic acid of MarA at position 89 is an inhibitor of MarA binding to many marboxes.
In an effort to understand why E89 is inhibitory, we examined the sequences of the 14 marboxes for which we have data (Table ). We noticed that five have a T at position 12 and that the binding to none of these sequences is increased by the E89A substitution (less than 1.7-fold). In contrast, of the remaining nine sequences that do not have a T at this position, the binding of E89A was increased for six of them by >2.5-fold and for the seventh by 1.7-fold; only two do not show increased binding. Although the cocrystal structure of MarA with the marRAB
marbox DNA (with a T at position 12) indicates no interaction between the two molecules at this position (36
), we considered the possibility that steric hindrance between the marbox DNA and MarA could limit activation by MarA when position 12 is not a T residue (see the Discussion for a fuller treatment).
To examine one facet of this possibility, namely, that the glutamic acid side chain of E89 sterically inhibits interaction with marbox DNAs lacking a T at position 12, we tested the effects of several amino acid substitutions at residue 89 on the fpr::lacZ fusion and the mdtG::lacZ fusion, the two fusions that showed the greatest effects of E89A on activation (see above). Plasmids carrying WT MarA, the MarA variant MarA(E89A) (with a nonpolar single methyl group side chain), MarA(E89G) (with no side chain), MarA(E89D) (with a side chain one methylene group shorter than glutamic acid), MarA(E89V) (with the amino acid present at the corresponding position of SoxS and having a dimethyl methylene side chain,), WT SoxS, and SoxS(V83E) were introduced into these fusions and assayed for β-galactosidase. Again (Table ), activation of these promoters by SoxS was much greater than that by MarA (Fig. ). Similarly, MarA(E89A) was considerably more active than WT MarA. In contrast, the E89D variant was less active than WT MarA for fpr and completely inactive (indistinguishable from the control plasmid) for mdtG. E89G was approximately twice as active as WT MarA for fpr and 4-fold more active for mdtG although for both promoters it was considerably less active than E89A. The E89V variant was marginally more active than the WT for both promoters but substantially less so than E89A. SoxS(V83E) reduced the activation of these promoters relative to WT SoxS, but SoxS(V83E) was still more active than WT MarA. (The activations shown here are greater than those apparent in Table because the expression of the plasmids carrying MarA, SoxS, and their mutants is not entirely shut off by lacIq so that the increases expressed in Table ppear smaller.) As outlined in the Discussion, these results, namely, (i) that variant E89D is virtually inactive, (ii) that E89G is very active although to a lesser extent than E89A, (iii) that E89V is only marginally more active than the WT, and (iv) that SoxS(V83E) has reduced activation although not to the low levels expressed by WT MarA, are consistent with the possibility that that the side chain of E89 sterically inhibits interaction with the DNA for marbox sequences lacking a T at position 12.
FIG. 3. Activation of class I promoters by MarA and SoxS and their mutants at position E89 (MarA) or V83 (SoxS). The absolute β-galactosidase values (Miller units [MU]) for the fpr::lacZ fusion (left-hand scale) and the mdtG::lacZ and acrAB::lacZ fusions (more ...)
If, as outlined above and presented in greater detail in the Discussion, steric interference with the phosphate between positions 12 and 13 and the glutamic acid side chain at position 89 is responsible for the very poor activation of promoters such as fpr, then it would be predicted that binding of WT MarA to the fpr marbox would be enhanced if that phosphate were absent. We therefore compared the binding affinity of WT MarA to either a 36-bp double-stranded DNA containing the marbox sequence of fpr or with dsDNA of the same sequence and length but prepared so that the phosphate linkage between positions 12 and 13 of the marbox was eliminated (see Materials and Methods). Again (Fig. nd Table ), MarA bound very poorly to the fpr marbox (Fig. ). However, binding increased significantly when the fpr DNA lacked the phosphate group between positions 12 and 13 (Fig. ). In contrast, SoxS bound more tightly to dsDNA than to the discontinuous DNA (Fig. ). When the phosphate located 3 nt farther upstream (between positions 9 and 10) was absent, no significant alteration in binding was observed (Fig. ) although a small increase was observed when the phosphate between positions 15 and 16 was absent. We conclude that the phosphate group between nt 12 and 13 of the consensus sequence inhibits the ability of MarA to bind.
FIG. 4. Autoradiographs of gel retardation assays as in Fig. except that the DNA fragment was 36 nt long and corresponds to the 7 nt upstream and 9 nt downstream of the native fpr marbox (GGACTGGAAGGCTCAATCGATCAAATCAATCAGAGG; the marbox is in boldface). (more ...) Activation and marbox binding by MarA(Q91A).
The only other MarA mutation found here to differ significantly from WT MarA in the activation of class I promoters was Q91A (Table ). Of the seven class I promoters examined, MarA(Q91A) activated the acrAB, marRAB, and zwf promoters to similar extents as WT MarA but activated acnA, fpr, mdtG, and poxB to only 60% or less of MarA WT levels (Table ; see also below).
For the nine class II promoters, MarA(Q91A) significantly reduced the activation of fumC
, and ybjC
but had no significant effect on nfsB
, or yhbW.
MarA- (Q91) has been identified as forming van der Waals interactions with the methyl groups of the two thymidines that are complementary to the adenines at positions 17 and 18 of the consensus sequence (36
). Thus, it would be expected that the Q91A substitution might reduce activation of the seven class II promoters that have at least one A at position 17 or 18 but not the two promoters, nfsB
, that have no A residues at these positions. Indeed, nfsB
are among the promoters that Q91A activated to the same extent as WT MarA. Since Q91A reduced the expression of four of the seven class I promoters and four of the nine class II promoters, our results are inconsistent with the proposition that Q91 is principally required for interactions at class II promoters, as has been proposed for the analogous site (Q85) in SoxS (16
For the majority of these promoters, the gel mobility assay for binding of MarA to marboxes was insufficiently precise to determine whether there is a correlation between binding and activation by MarA(Q91A) relative to MarA (Table ). Among the class I promoters that exhibited measurable binding to MarA, MarA(Q91A) showed no greater binding or activation for acrAB
, a modest reduction in activation and binding for mdtG
, and a reduction in binding but not in activation for marRAB
. A modest reduction in binding with a small increase in activation was seen for the class II mdaB
promoter. Although not observed in these experiments, a more detailed analysis of the binding of MarA(Q91A) to the micF
marbox (using protein without the His6
tag) showed a very small reduction in binding concomitant with the reduced ability of MarA(Q91A) to activate the class II micF
). Thus, relative to MarA, there may be a correlation between activation and binding for Q91A at class I promoters, but none is obvious with regard to the class II promoters.
Activation of superoxide resistance by MarA(E89A).
If the glutamic acid at position 89 of MarA is a major determinant in vivo of the reduced activity of MarA at many promoters where SoxS is more active, we would expect cells carrying MarA(E89A)to be more resistant to superoxides than cells carrying WT MarA. This was tested with gradient plate assays of sensitivity to two superoxide-generating compounds, phenazine methosulfate (PMS) and menadione (Table ). Cells constitutively expressing MarA(E89A)were more resistant than WT MarA to PMS (1.7-fold) and, to a lesser extent, to menadione (1.3-fold). Comparable MICs with SoxS for PMS were 1.6-fold and for menadione 2.0-fold. Clearly, MarA(E89A)activates superoxide resistance to a greater extent than WT MarA. Curiously, the Q91A substitution had no effect on resistance to the superoxide generator PMS (MIC of 35 μM for both the Q91A mutant and the WT MarA) but lowered resistance to the superoxide generator menadione (MIC of 0.9 mM for the Q91A mutant and 1.8 mM for the WT).
Superoxide resistance of strains carrying MarA, MarA mutations, or SoxS