Richard E. Wolf

Proteolytic degradation of Escherichia coli transcription activators SoxS and MarA as the mechanism for reversing the induction of the superoxide (SoxRS) and multiple antibiotic resistance (Mar) regulons

In Escherichia coli, the SoxRS regulon confers resistance to redox‐cycling compounds, and the Mar regulon provides a defence against multiple antibiotics. The response regulators, SoxS and MarA, are synthesized de novo in response to their inducing signals and directly activate transcription of a common set of target genes. Although the mechanisms of transcription activation by SoxS and MarA have been well studied, little is known about how the systems are shut‐off once the inducing stress has subsided, except that de novo synthesis of the regulators is known to cease almost immediately. Here, we induced the SoxRS regulon and determined that, upon removal of the inducer, expression of the regulons genes quickly returns to the preinduced level. This rapid shut‐off indicates that the system is reset by an active process. We found that SoxS is unstable and infer that SoxS degradation is responsible for the rapid return of the system to the ground state upon removal of the inducing signal. We also found that MarA is unstable and that the instability of both proteins is intrinsic and unregulated. We used null mutations of protease genes to identify the proteases involved in the degradation of SoxS and MarA. Among single protease mutations, only lon mutations increased the half‐life of SoxS and MarA. In addition, SoxS appeared to be nearly completely stable in a lon ftsH double mutant. Using hexahistidine tags placed at the respective ends of the activators, we found that access to the amino‐terminus is essential for the proteolytic degradation.

Ambidextrous transcriptional activation by SoxS: requirement for the C-terminal domain of the RNA polymerase alpha subunit in a subset of Escherichia coli superoxide-inducible genes

Purified MalE–SoxS fusion protein specifically stimulated in vitro transcription of the Escherichia colizwffprfumCmicFnfo, and sodA genes, indicating that activation of the superoxide regulon requires only SoxS. As in vivo, a 21 bp sequence adjacent to the zwf promoter was able to activate transcription of an heterologous promoter in vitro. Activation of zwf and fpr transcription required the C‐terminal domain (CTD) of the RNA polymerase alpha subunit, while stimulation of fumCmicFnfo, and sodA transcription was independent of CTD truncation. Thus, like the catabolite gene activator protein (CAP), SoxS is an ‘ambidextrous’ activator, activation only requiring the α CTD in a subset of regulated promoters. Indeed, the −35 hexamers of the zwf and fpr promoters lie downstream of the respective MalE–SoxS binding sites, while the binding sites of fumCmicFnfo, and sodA overlap their −35 promoter hexamers.

Interdependence of the position and orientation of SoxS binding sites in the transcriptional activation of the class I subset of Escherichia coli superoxide-inducible promoters

SoxS is the direct transcriptional activator of the member genes of the Escherichia coli superoxide regulon. At class I SoxS‐dependent promoters, e.g. zwf and fpr, whose SoxS binding sites (‘soxbox’) lie upstream of the −35 region of the promoter, activation requires the C‐terminal domain of the RNA polymerase α‐subunit, while at class II SoxS‐dependent promoters, e.g. fumC and micF, whose binding sites overlap the −35 region, activation is independent of the α‐CTD. To determine whether SoxS activation of its class I promoters shows the same helical phase‐dependent spacing requirement as class I promoters activated by catabolite gene activator protein, we increased the 7 bp distance between the 20 bp zwf soxbox and the zwf−35 promoter hexamer by 5 bp and 11 bp, and we decreased the 15 bp distance between the 20 bp fpr soxbox and the fpr−35 promoter hexamer by the same amounts. In both cases, displacement of the binding site by a half or full turn of the DNA helix prevented transcriptional activation. With constructs containing the binding site of one gene fused to the promoter of the other, we demonstrated that the positional requirements are a function of the specific binding site, not the promoter. Supposing that opposite orientation of the SoxS binding site at the two promoters might account for the positional requirements, we placed the zwf and fpr soxboxes in the reverse orientation at the various positions upstream of the promoters and determined the effect of orientation on transcription activation. We found that reversing the orientation of the zwf binding site converts its positional requirement to that of the fpr binding site in its normal orientation, and vice versa. Analysis by molecular information theory of DNA sequences known to bind SoxS in vitro is consistent with the opposite orientation of the zwf and fpr soxboxes.

Systematic mutagenesis of the DNA binding sites for SoxS in the Escherichia coli zwf and fpr promoters: identifying nucleotides required for DNA binding and transcription activation

SoxS is the direct transcriptional activator of at least 15 genes of the Escherichia coli superoxide regulon. SoxS is small (107 amino acids), binds DNA as a monomer and recognizes a highly degenerate DNA binding site, termed ‘soxbox’. Like other members of the AraC/XylS family, SoxS has two putative helix–turn–helix (HTH) DNA‐binding motifs, and it has been proposed that each HTH motif recognizes a highly conserved recognition element of the soxbox. To determine which nucleotides are important for SoxS binding, we conducted a systematic mutagenesis of the DNA binding sites for SoxS in the zwf and fpr promoters and determined the effect of the soxbox mutations on SoxS DNA binding and transcription activation in vivo by measuring β‐galactosidase activity in strains with fusions to lacZ. We found that the sequences GCAC and CAAA, termed recognition elements 1 and 2 (RE 1 and RE 2), respectively, are critical for SoxS binding, as mutations within these elements severely hinder or eliminate SoxS‐dependent transcription activation; substitutions within RE 2 (CAAA), however, are tolerated better than changes within RE 1 (GCAC). Although substitutions at the seven positions separating the two REs had only a modest effect on SoxS binding, AT basepairs were favoured within this ‘spacer’ region, presumably because, by facilitating DNA bending, they help bring the two recognition elements into proper juxtaposition. We also found that the ‘invariant A’ present at position 1 of 14/15 functional soxboxes identified thus far is important for SoxS binding, as a change to any other nucleotide at this position reduced SoxS‐dependent transcription by ≈ 50%. In addition, positions surrounding the REs seem to show a context effect, in that certain substitutions there have little or no effect when the RE has the optimal binding sequence, but produce a pronounced effect when the RE has a suboptimal sequence. We propose that these nucleotides play an important role in effecting differential expression from the various promoters. Lastly, we used gel retardation assays to show that alterations in transcription activation in vivo are caused by effects on DNA binding. Based on this exhaustive mutagenesis, we propose the following optimal sequence for SoxS binding: AnVGCACWWWnKRHCAAAHn (n = A, C, G, T; V = A, C, G; W = A, T; K = G, T; R = A, G; H = A, C, T).

Purification of a MaIE‐SoxS fusion protein and identification of the control sites of Escherichia coli superoxide‐inducible genes

In Escherichia coli, the soxRS genes effect the cells defence against superoxide by activating the transcription of more than 14 genes, including zwf, sodA, nfo, micF and fumC. Previous work from other laboratories has Indicated that SoxR is the sensor of oxidative stress and induces synthesis of SoxS, which in turn activates transcription of the regulons target genes. Although SoxS appears to be a DNA‐binding protein, its ability to bind to the promoter regions of target genes has not been demonstrated. To facilitate purification and characterization of SoxS, we constructed a fusion of soxS to MalE, which encodes maltose‐binding protein, and demonstrated that the in vivo expression of the MaIE‐SoxS fusion protein can provide SoxS function to a soxRS deletion mutant. We purified the fusion protein by affinity chromatography on an amylose column. The purified fusion protein stimulated m vitro expression of zwf in a coupled transcription‐translation system and formed specific complexes with DNA fragments carrying target gene promoters. Moreover, MalE–SoxS protected from DNase I attack 22–27 bp segments immediately adjacent to or overlapping the −35 hexamers of the zwf, sodA, nfo, micF. and fumC promoters. The protected regions revealed a consensus ‘soxbox’ sequence.

Inhibition of Lon-dependent degradation of the Escherichia coli transcription activator SoxS by interaction with 'soxbox' DNA or RNA polymerase

Escherichia coli SoxS, the direct transcription activator of the SoxRS (superoxide) regulon, is intrinsically unstable with an in vivo half‐life of ∼2 min. Overexpression of SoxS is lethal, but mutations interfering with DNA binding relieve the toxicity. Here, we determined the effects on the half‐life of SoxS of alanine substitutions that confer defects in positive control, i.e. transcription activation, or in specific DNA binding. We found that both types of mutations render SoxS more unstable than the wild‐type protein, as if ‘soxbox’ DNA and RNA polymerase serve as stabilizing ligands in vivo that protect SoxS from degradation by Lon, the protease shown previously to be primarily responsible for its turnover. Indeed, we found that the addition of soxbox DNA or RNA polymerase to an in vitro degradation system decreases the rate of SoxS proteolysis by Lon protease. To the best of our knowledge, these are the first examples of target DNA and RNA polymerase serving as ligands that inhibit the turnover of an unstable transcription activator.

Transcription Activation by a Variety of AraC/XylS Family Activators Does Not Depend on the Class II-Specific Activation Determinant in the N-Terminal Domain of the RNA Polymerase Alpha Subunit

The N-terminal domain of the RNA polymerase alpha subunit (alpha-NTD) was tested for a role in transcription activation by a variety of AraC/XylS family members. Based on substitutions at residues 162 to 165 and an extensive genetic screen we conclude that alpha-NTD is not an activation target for these activators.

Characterization of TetD as a transcriptional activator of a subset of genes of the Escherichia coli SoxS/MarA/Rob regulon

In Escherichia coli, SoxS, MarA and Rob form a closely related subset of the AraC/XylS family of positive regulators, sharing ∼42% amino acid sequence identity over the length of SoxS and the ability to activate transcription of a common set of target genes that provide resistance to redox‐cycling compounds and antibiotics. On the basis of its ∼43% amino acid sequence identity with SoxS, MarA and Rob, TetD, encoded by transposon Tn10, appears to be a fourth member of the subset. However, although its expression has been shown to be negatively regulated by TetC and not inducible by tetracycline, the physiological function of TetD is unknown. Accordingly, in the work presented here, we initiate a molecular characterization of TetD. We show that expression of TetD activates transcription of a subset of the SoxS/MarA/Rob regulon genes and confers resistance to redox‐cycling compounds and antibiotics. We show that mutations in the putative TetD binding site of a TetD‐activatable promoter and a mutation in the proteins N‐terminal DNA recognition helix interfere with transcription activation, thereby indicating that TetD directly activates target gene transcription. Finally, we show that TetD, like SoxS and MarA, is intrinsically unstable; however, unlike SoxS and MarA, TetD is not degraded by Lon or any of the cells known cytoplasmic ATP‐dependent proteases. Thus, we conclude that TetD is a bona fide member of the SoxS/MarA/Rob subfamily of positive regulators.

Transcription Activation by Escherichia coli Rob at Class II Promoters: Protein–Protein Interactions between Rob’s N-Terminal Domain and the σ70 Subunit of RNA Polymerase

Bacterial transcription activators regulate transcription by making essential protein-protein interactions with RNA polymerase, for example, with region 4 of the σ(70) subunit (σ(70) R4). Rob, SoxS, and MarA comprise a closely related subset of members of the AraC/XylS family of transcription factors that activate transcription of both class I and class II promoters. Recently, we showed that interactions between SoxS and σ(70) R4 occlude the binding of σ(70) R4 to the -35 promoter element of class II promoters. Although Rob shares many similarities with SoxS, it contains a C-terminal domain (CTD) that the other paralogs do not. Thus, a goal of this study was to determine whether Rob makes protein-protein interactions with σ(70) R4 at class II promoters and, if so, whether the interactions occlude the binding of σ(70) R4 to the -35 hexamer despite the presence of the CTD. We found that although Rob makes fewer interactions with σ(70) R4 than SoxS, the two proteins make the same, unusual, position-dependent interactions. Importantly, we found that Rob occludes σ(70) R4 from binding the -35 hexamer, just as does SoxS. Thus, the CTD does not substantially alter the way Rob interacts with σ(70) R4 at class II promoters. Moreover, in contrast to inferences drawn from the co-crystal structure of Rob bound to robbox DNA, which showed that only one of Robs dual helix-turn-helix (HTH) DNA binding motifs binds a recognition element of the promoters robbox, we determined that the two HTH motifs each bind a recognition element in vivo.