A dual role in regulation and toxicity for the disordered N-terminus of the toxin GraT

Abstract

Bacterial toxin-antitoxin (TA) modules are tightly regulated to maintain growth in favorable conditions or growth arrest during stress. A typical regulatory strategy involves the antitoxin binding and repressing its own promoter while the toxin often acts as a co-repressor. Here we show that Pseudomonas putida graTA -encoded antitoxin GraA and toxin GraT differ from other TA proteins in the sense that not the antitoxin but the toxin possesses a flexible region. GraA auto-represses the graTA promoter: two GraA dimers bind cooperatively at opposite sides of the operator sequence. Contrary to other TA modules, GraT is a de-repressor of the graTA promoter as its N-terminal disordered segment prevents the binding of the GraT 2 A 2 complex to the operator. Removal of this region restores operator binding and abrogates Gr aT toxicity. GraTA represents a TA module where a flexible region in the toxin rather than in the antitoxin controls operon expression and toxin activity.

Type II Toxin-Antitoxin Systems: Evolution and Revolutions

“…3D). Antitoxins of reverse-organized systems from other species were also shown to lose affinity for their operators upon toxin binding, i.e., GraA from Pseudomonas putida and HigA from Proteus vulgaris (100,101). Therefore, while conditional cooperativity is supposedly the prevalent mode of TA autoregulation, other modes of regulation might exist, especially for systems in "reverse" configuration as described above.…”

Pseudomonas putidachromosomal toxin-antitoxin systems carry neither clear fitness benefits nor big costs

“…In order to analyze the benefits and costs of multitude TA systems, the 13 TA loci were deleted from P. putida PaW85 (isogenic to KT2440) chromosome in the following order: graTA (PP_1586-1585), res-xre (PP_2433-2434), higBA (PP_1199-1198), hicAB-1 (PP_1480-1479), relE 2 -higA 2 (PP_5435-PP_0274), mqsRA (PP_4205-4204), mazEF (PP_0770-0771), PP_1716-1717, brnTA (PP_4530-4529), PP_4151-4152, relBE (PP_1268-1267), yefM-yoeB (PP_2940-2939) and relB 2 -parE (PP_2499-2500) (Table 1). The functionality of only four ( graTA, res-xre, mqsRA and mazEF ) out of these 13 TA modules has been studied experimentally 28,38,39,41-43 , while the other loci are putative TA pairs predicted in silico only 36 . TADB2 database annotates two more putative TA loci, the hicAB-2 (PP_3900-3899) and PP_3032-3033, which are located in prophage 1 and prophage 2 genomes, respectively 36,44 .…”

“…The most thoroughly studied is HigBA family GraTA module, which codes for an unusually stable antitoxin GraA 37 and a ribosome-dependent mRNase GraT characterized by conditional toxicity 28,38 . Deletion of antitoxin gene graA from P. putida chromosome affects bacterial growth at 30 °C only slightly, but causes severe growth and ribosome biogenesis defect at lower temperatures 28,38-40 . GraT has ability to affect stress tolerance both positively and negatively, yet deletion of the graTA operon has no effect on P. putida fitness 28 .…”

1H, 13C, and 15N backbone and side chain chemical shift assignment of YdaS, a monomeric member of the HigA family

“…Compared to GraA and other HigA proteins, the presence of a C-terminal intrinsically disordered region is unique for YdaS. While this is a common feature of many TA antitoxins, the HigA family represented by Proteus vulgaris HigA and Pseudomonas putida GraA does not carry such a region (Schureck et al 2014;Talavera et al 2019). Therefore, YdaS appears to represent a novel family within the HigA superfamily, although it is not directly part of a toxin-antitoxin Fig.…”

“…The first type, represented by Vibrio cholerae HigA, contains a folded C-terminal regulatory helix-turn-helix domain that is preceded by an intrinsically disordered N-terminal domain responsible for toxin neutralization (Hadži et al 2017). The second type, represented by HigA proteins from Proteus vulgaris plasmid Rts1 (Schureck et al 2014) and Pseudomonas putida (Talavera et al 2019), is a fully folded single helixturn-helix domain protein that combines both DNA binding and toxin-neutralizing functions. The third type, as exemplified by members found in E. coli and Shigella flexneri, has two distinct globular domains: an N-terminal dimerization domain and a C-terminal HTH domain that are connected by a long α-helix (Yang et al 2016).…”