Regulation of the phd/doc toxin-antitoxin operon involves the toxin Doc as co- or derepressor depending on the ratio between Phd and Doc, a phenomenon known as conditional cooperativity. The mechanism underlying this observed behavior is not understood. Here we show that monomeric Doc engages two Phd dimers on two unrelated binding sites. The binding of Doc to the intrinsically disordered C-terminal domain of Phd structures its N-terminal DNA-binding domain, illustrating allosteric coupling between highly disordered and highly unstable domains. This allosteric effect also couples Doc neutralization to the conditional regulation of transcription. In this way, higher levels of Doc tighten repression up to a point where the accumulation of toxin triggers the production of Phd to counteract its action. Our experiments provide the basis for understanding the mechanism of conditional cooperative regulation of transcription typical of toxin-antitoxin modules. This model may be applicable for the regulation of other biological systems.

Toxin-antitoxin modules are small regulatory circuits that ensure survival of bacterial populations under challenging environmental conditions. The ccd toxin-antitoxin module on the F plasmid codes for the toxin CcdB and its antitoxin CcdA. CcdB poisons gyrase while CcdA actively dissociates CcdB:gyrase complexes in a process called rejuvenation. The CcdA:CcdB ratio modulates autorepression of the ccd operon. The mechanisms behind both rejuvenation and regulation of expression are poorly understood. We show that CcdA binds consecutively to two partially overlapping sites on CcdB, which differ in affinity by six orders of magnitude. The first, picomolar affinity interaction triggers a conformational change in CcdB that initiates the dissociation of CcdB:gyrase complexes by an allosteric segmental binding mechanism. The second, micromolar affinity binding event regulates expression of the ccd operon. Both functions of CcdA, rejuvenation and autoregulation, are mechanistically intertwined and depend crucially on the intrinsically disordered nature of the CcdA C-terminal domain.

Most bacteria control oxidative stress through the H2O2-responsive transactivator OxyR, a member of the LTTR family (LysR Type Transcriptional Regulators), which activates the expression of defensive genes such as those encoding catalases, alkyl hydroperoxide reductases and superoxide dismutases. In the human opportunistic pathogen Pseudomonas aeruginosa, OxyR positively regulates expression of the oxidative stress response genes katA, katB, ahpB and ahpCF. To identify additional targets of OxyR in P. aeruginosa PAO1, we performed chromatin immunoprecipitation in combination with whole genome tiling array analyses (ChIP-chip). We detected 56 genes including all the previously identified defensive genes and a battery of novel direct targets of OxyR. Electrophoretic mobility shift assays (EMSAs) for selected newly identified targets indicated that ∼70% of those were bound by purified oxidized OxyR and their regulation was confirmed by quantitative real-time polymerase chain reaction. Furthermore, a thioredoxin system was identified to enzymatically reduce OxyR under oxidative stress. Functional classification analysis showed that OxyR controls a core regulon of oxidative stress defensive genes, and other genes involved in regulation of iron homeostasis (pvdS), quorum-sensing (rsaL), protein synthesis (rpsL) and oxidative phosphorylation (cyoA and snr1). Collectively, our results indicate that OxyR is involved in oxidative stress defense and regulates other aspects of cellular metabolism as well.

The carAB operon of Escherichia coli K-12, which encodes the two subunits of carbamoyl-phosphate synthetase (glutamine hydrolyzing) [carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating); EC 6.3.5.5], is cumulatively repressed by arginine and the pyrimidines. We describe the structure of the control region of carAB and the sequence of the carA gene. Nuclease S1 mapping experiments show that two adjacent tandem promoters within the carAB control region serve as initiation sites. The upstream promoter P1 is controlled by pyrimidines; the downstream promoter P2 is regulated by arginine. Attenuation control does not appear to be involved in the expression of carAB. A possible mechanism by which control at these promoters concurs to produce a cumulative pattern of repression is discussed. The translational start of carA is atypical; it consists of a UUG or AUU codon.

SummarySs-LrpB, a novel Lrp-like DNA-binding protein from the hyperthermophilic crenarchaeon Sulfolobus solfataricus , was shown to bind cooperatively to three regularly spaced targets in its own control region, with as consensus the 15 bp palindrome 5 ¢ ¢ ¢ ¢ -TTGYAW WWWWTRCAA-3 ¢ ¢ ¢ ¢ . Binding to the border sites occurred with high affinity; the target in the middle proved to be a low affinity site which is stably bound only when both flanking sites are occupied. Ss-LrpB contacts two major groove segments and the intervening minor groove of each site, all aligned on one face of the helix. The operator shows intrinsic bending and is increasingly deformed upon binding of SsLrpB to one, two and three targets. Complex formation relies therefore on DNA conformability, protein-DNA and protein-protein contacts. Mobility-shift assays and in gel footprinting indicate that Ss-LrpB and the transcription factors TATA-box binding protein (TBP) and transcription factor B (TFB) can bind simultaneously to the control region. Based on these findings we present a model for the construction of the higher order nucleoprotein complexes and a hypothesis for the autoregulatory process. The latter is based on the concentration-dependent formation of distinct complexes exhibiting different stoichiometries and conformations, which could positively and negatively affect promoter activity.