Complex interplay between the LysR‐type regulator AtzR and its binding site mediates atzDEF activation in response to two distinct signals

Abstract

SummaryAtzR is a LysR-type regulator responsible for activation of the cyanuric acid utilization operon atzDEF. AtzR binds the PatzDEF promoter region at a strong recognition element, designated the repressor binding site, and a weaker binding determinant, the activator binding site (ABS). AtzR activates transcription in response to two dissimilar signals, nitrogen limitation and cyanuric acid. In the present work we analyse the structure and function of the cis-acting elements involved in AtzR activation of atzDEF. Hydroxyl radical footprinting assays revealed that the ABS is composed of three functional subsites spaced at one helix-turn intervals. Two modes of interaction with the ABS are detected in vitro: AtzR binds at the ABS-2 and ABS-3 subsites in the absence of inducer, and relocates to interact with the ABS-1 and ABS-2 subsites in the presence of cyanuric acid. In vivo mutational analysis indicates that ABS-1 and ABS-2 are required for full PatzDEF activation in all conditions. In contrast, ABS-3 acts as a 'subunit trap' that hinders productive AtzR interactions with ABS-1 and ABS-2. Our results strongly suggest an activation model in which cyanuric acid and nitrogen limitation cooperate to reposition AtzR from an inactive, ABS-3 bound configuration to an active, ABS-1-and ABS-2-bound configuration.

Transcriptional Organization and Regulatory Elements of a Pseudomonas sp. Strain ADP Operon Encoding a LysR-Type Regulator and a Putative Solute Transport System

“…In addition, hypersensitive positions were noted at Ϫ139, Ϫ136, Ϫ129, Ϫ100, Ϫ99, Ϫ90, and Ϫ85 (bottom strand) and Ϫ130, Ϫ96, Ϫ95, Ϫ87, Ϫ86, and Ϫ85 (top strand). Continuous protection of the RBS and the presence of strongly hypersensitive positions downstream from the RBS were reminiscent of the AtzR interaction with the PatzDEF promoter region and suggested that AtzR bends DNA upon binding (40,42). However, unlike the PatzDEF promoter region, additional protected positions extending toward the PatzT promoter were not observed, suggesting that the interaction of AtzR with DNA outside the strong RBS element is likely very weak or absent.…”

“…Gel mobility shift assays with AtzR were performed essentially as described previously (42). Probes containing the wild-type and the mutant versions of the AtzR binding site (AtzR-L, AtzR-R, and AtzR-LR) were obtained by PCR using the corresponding lacZ fusion plasmids pMPO805, pMPO815, pMOPO816, and pMPO821 as templates and oligonucleotides Del-RBS and R-SalI as primers.…”

“…DNase I footprinting assays. DNase I footprinting assays with AtzR were performed essentially as described previously (42). Probes for DNase I footprinting were obtained by PCR amplification using pMPO805 as the template and primers delABS and fpIHF2 for the top strand and fpNtrC1 and fpIHF4 for the bottom strand.…”

“…In contrast, DNase I protection of the PatzT promoter region by AtzR does not extend far beyond the limits of the RBS. Sequence comparison revealed that the sequences of the three individual binding sites within the ABS, designated ABS-1, ABS-2, and ABS-3 (42), are poorly conserved in the PatzT promoter region. Due to this fact, interaction in this region may be too weak to be detected by means of DNase I footprinting.…”

MexT Functions as a Redox-Responsive Regulator Modulating Disulfide Stress Resistance in Pseudomonas aeruginosa

“…Several LTTRs are known to activate gene expression as higher oligomeric complexes, which form on recognition of their cognate signals (2,53,55,57). Studies on the classical LysR regulator OxyR, which forms oligomeric complexes when oxidized, suggest that a subset of LTTRs are activated via a redox-sensing mechanism (2,69,70,72).…”