The structure of DarB in complex with RelNTD reveals nonribosomal activation of Rel stringent factors.

Rel stringent factors are bifunctional ribosome-associated enzymes that catalyze both synthesis and hydrolysis of the alarmones (p)ppGpp. Besides the allosteric control by starved ribosomes and (p)ppGpp, Rel is regulated by various protein factors depending on specific stress conditions, including the c-di-AMP-binding protein DarB. However, how these effector proteins control Rel remains unknown. We have determined the crystal structure of the DarB2:RelNTD2 complex, uncovering that DarB directly engages the SYNTH domain of Rel to stimulate (p)ppGpp synthesis. This association with DarB promotes a SYNTH-primed conformation of the N-terminal domain region, markedly increasing the affinity of Rel for ATP while switching off the hydrolase activity of the enzyme. Binding to c-di-AMP rigidifies DarB, imposing an entropic penalty that precludes DarB-mediated control of Rel during normal growth. Our experiments provide the basis for understanding a previously unknown mechanism of allosteric regulation of Rel stringent factors independent of amino acid starvation.

Structure of SpoT reveals evolutionary tuning of catalysis via conformational constraint.

Stringent factors orchestrate bacterial cell reprogramming through increasing the level of the alarmones (p)ppGpp. In Beta- and Gammaproteobacteria, SpoT hydrolyzes (p)ppGpp to counteract the synthetase activity of RelA. However, structural information about how SpoT controls the levels of (p)ppGpp is missing. Here we present the crystal structure of the hydrolase-only SpoT from Acinetobacter baumannii and uncover the mechanism of intramolecular regulation of 'long'-stringent factors. In contrast to ribosome-associated Rel/RelA that adopt an elongated structure, SpoT assumes a compact τ-shaped structure in which the regulatory domains wrap around a Core subdomain that controls the conformational state of the enzyme. The Core is key to the specialization of long RelA-SpoT homologs toward either synthesis or hydrolysis: the short and structured Core of SpoT stabilizes the τ-state priming the hydrolase domain for (p)ppGpp hydrolysis, whereas the longer, more dynamic Core domain of RelA destabilizes the τ-state priming the monofunctional RelA for efficient (p)ppGpp synthesis.

RelA-SpoT Homolog toxins pyrophosphorylate the CCA end of tRNA to inhibit protein synthesis.

RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes.

The Disordered C-Terminus of the Chaperone DnaK Increases the Competitive Fitness of Pseudomonas putida and Facilitates the Toxicity of GraT.

Chaperone proteins are crucial for proper protein folding and quality control, especially when cells encounter stress caused by non-optimal temperatures. DnaK is one of such essential chaperones in bacteria. Although DnaK has been well characterized, the function of its intrinsically disordered C-terminus has remained enigmatic as the deletion of this region has been shown to either enhance or reduce its protein folding ability. We have shown previously that DnaK interacts with toxin GraT of the GraTA toxin-antitoxin system in Pseudomonas putida. Interestingly, the C-terminal truncation of DnaK was shown to alleviate GraT-caused growth defects. Here, we aim to clarify the importance of DnaK in GraT activity. We show that DnaK increases GraT toxicity, and particularly important is the negatively charged motif in the DnaK C-terminus. Given that GraT has an intrinsically disordered N-terminus, the assistance of DnaK is probably needed for re-modelling the toxin structure. We also demonstrate that the DnaK C-terminal negatively charged motif contributes to the competitive fitness of P. putida at both high and optimal growth temperatures. Thus, our data suggest that the disordered C-terminal end of DnaK enhances the chaperone functionality.

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

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 GraT2A2 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.

Pseudomonas putida Responds to the Toxin GraT by Inducing Ribosome Biogenesis Factors and Repressing TCA Cycle Enzymes.

The potentially self-poisonous toxin-antitoxin modules are widespread in bacterial chromosomes, but despite extensive studies, their biological importance remains poorly understood. Here, we used whole-cell proteomics to study the cellular effects of the Pseudomonas putida toxin GraT that is known to inhibit growth and ribosome maturation in a cold-dependent manner when the graA antitoxin gene is deleted from the genome. Proteomic analysis of P. putida wild-type and ΔgraA strains at 30 °C and 25 °C, where the growth is differently affected by GraT, revealed two major responses to GraT at both temperatures. First, ribosome biogenesis factors, including the RNA helicase DeaD and RNase III, are upregulated in ΔgraA. This likely serves to alleviate the ribosome biogenesis defect of the ΔgraA strain. Secondly, proteome data indicated that GraT induces downregulation of central carbon metabolism, as suggested by the decreased levels of TCA cycle enzymes isocitrate dehydrogenase Idh, α-ketoglutarate dehydrogenase subunit SucA, and succinate-CoA ligase subunit SucD. Metabolomic analysis revealed remarkable GraT-dependent accumulation of oxaloacetate at 25 °C and a reduced amount of malate, another TCA intermediate. The accumulation of oxaloacetate is likely due to decreased flux through the TCA cycle but also indicates inhibition of anabolic pathways in GraT-affected bacteria. Thus, proteomic and metabolomic analysis of the ΔgraA strain revealed that GraT-mediated stress triggers several responses that reprogram the cell physiology to alleviate the GraT-caused damage.

Production, biophysical characterization and crystallization of Pseudomonas putida GraA and its complexes with GraT and the graTA operator.

The graTA operon from Pseudomonas putida encodes a toxin-antitoxin module with an unusually moderate toxin. Here, the production, SAXS analysis and crystallization of the antitoxin GraA, the GraTA complex and the complex of GraA with a 33 bp operator fragment are reported. GraA forms a homodimer in solution and crystallizes in space group P21, with unit-cell parameters a = 66.9, b = 48.9, c = 62.7 Å, ß = 92.6°. The crystals are likely to contain two GraA dimers in the asymmetric unit and diffract to 1.9 Šresolution. The GraTA complex forms a heterotetramer in solution. Crystals of the GraTA complex diffracted to 2.2 Šresolution and are most likely to contain a single heterotetrameric GraTA complex in the asymmetric unit. They belong to space group P41 or P43, with unit-cell parameters a = b = 56.0, c = 128.2 Å. The GraA-operator complex consists of a 33 bp operator region that binds two GraA dimers. It crystallizes in space group P31 or P32, with unit-cell parameters a = b = 105.6, c = 149.9 Å. These crystals diffract to 3.8 Šresolution.

The toxin GraT inhibits ribosome biogenesis.

Most bacteria encode numerous chromosomal toxin-antitoxin (TA) systems that are proposed to contribute to stress tolerance, as they are able to shift the cells to a dormant state. Toxins act on a variety of targets with the majority attacking the translational apparatus. Intriguingly, the toxicity mechanisms of even closely related toxins may differ essentially. Here, we report on a new type of TA toxin that inhibits ribosome biogenesis. GraT of the GraTA system has previously been described in Pseudomonas putida as an unusually moderate toxin at optimal growth temperatures. However, GraT causes a severe growth defect at lower temperatures. Here, we demonstrate that GraT causes the accumulation of free ribosomal subunits. Mapping the rRNA 5' ends reveals incomplete processing of the free subunits and quantification of modified nucleosides shows an underrepresentation of late subunit assembly specific modifications. This indicates that GraT inhibits ribosome subunit assembly. Interestingly, GraT effects can be alleviated by modification of the chaperone DnaK, a known facilitator of late stages in ribosome biogenesis. We show that GraT directly interacts with DnaK and suggest two possible models for the role of this interaction in GraT toxicity.

Stability of the GraA Antitoxin Depends on Growth Phase, ATP Level, and Global Regulator MexT.

UNLABELLED: Bacterial type II toxin-antitoxin systems consist of a potentially poisonous toxin and an antitoxin that inactivates the toxic protein by binding to it. Most of the toxins regulate stress survival, but their activation depends on the stability of the antitoxin that has to be degraded in order for the toxin to be able to attack its cellular targets. The degradation of antitoxins is usually rapid and carried out by ATP-dependent protease Lon or Clp, which is activated under stress conditions. The graTA system of Pseudomonas putida encodes the toxin GraT, which can affect the growth rate and stress tolerance of bacteria but is under most conditions inactivated by the unusually stable antitoxin GraA. Here, we aimed to describe the stability features of the antitoxin GraA by analyzing its degradation rate in total cell lysates of P. putida. We show that the degradation rate of GraA depends on the growth phase of bacteria being fastest in the transition from exponential to stationary phase. In accordance with this, higher ATP levels were shown to stabilize GraA. Differently from other antitoxins, the main cellular proteases Lon and Clp are not involved in GraA stability. Instead, GraA seems to be degraded through a unique pathway involving an endoprotease that cleaves the antitoxin into two unequal parts. We also identified the global transcriptional regulator MexT as a factor for destabilization of GraA, which indicates that the degradation of GraA may be induced by conditions similar to those that activate MexT. IMPORTANCE: Toxin-antitoxin (TA) modules are widespread in bacterial chromosomes and have important roles in stress tolerance. As activation of a type II toxin is triggered by proteolytic degradation of the antitoxin, knowledge about the regulation of the antitoxin stability is critical for understanding the activation of a particular TA module. Here, we report on the unusual degradation pathway of the antitoxin GraA of the recently characterized GraTA system. While GraA is uncommonly stable in the exponential and late-stationary phases, its degradation increases in the transition phase. The degradation pathway of GraA involves neither Lon nor Clp, which usually targets antitoxins, but rather an unknown endoprotease and the global regulator MexT, suggesting a new type of regulation of antitoxin stability.

A moderate toxin, GraT, modulates growth rate and stress tolerance of Pseudomonas putida.

Chromosomal toxin-antitoxin (TA) systems are widespread among free-living bacteria and are supposedly involved in stress tolerance. Here, we report the first TA system identified in the soil bacterium Pseudomonas putida. The system, encoded by the loci PP1586-PP1585, is conserved in pseudomonads and belongs to the HigBA family. The new TA pair was named GraTA for the growth rate-affecting ability of GraT and the antidote activity of GraA. The GraTA system shares many features common to previously described type II TA systems. The overexpression of GraT is toxic to the antitoxin deletion mutants, since the toxin's neutralization is achieved by binding of the antitoxin. Also, the graTA operon structure and autoregulation by antitoxin resemble those of other TA loci. However, we were able to delete the antitoxin gene from the chromosome, which shows the unusually mild toxicity of innate GraT compared to previously described toxins. Furthermore, GraT is a temperature-dependent toxin, as its growth-regulating effect becomes more evident at lower temperatures. Besides affecting the growth rate, GraT also increases membrane permeability, resulting in higher sensitivity to some chemicals, e.g., NaCl and paraquat. Nevertheless, the active toxin helps the bacteria survive under different stressful conditions and increases their tolerance to several antibiotics, including streptomycin, kanamycin, and ciprofloxacin. Therefore, our data suggest that GraT may represent a new class of mild chromosomal regulatory toxins that have evolved to be less harmful to their host bacterium. Their moderate toxicity might allow finer growth and metabolism regulation than is possible with strong growth-arresting or bactericidal toxins.

The ColRS system is essential for the hunger response of glucose-growing Pseudomonas putida.

BACKGROUND: The survival of bacteria largely depends on signaling systems that coordinate cell responses to environmental cues. Previous studies on the two-component ColRS signal system in Pseudomonas putida revealed a peculiar subpopulation lysis phenotype of colR mutant that grows on solid glucose medium. Here, we aimed to clarify the reasons for the lysis of bacteria. RESULTS: We present evidence that the lysis defect of P. putida colR mutant is linked to hunger response. A subpopulation prone to lysis was located in the periphery of bacterial cultures growing on solid medium. Cell lysis was observed in glucose-limiting, but not in glucose-rich conditions. Furthermore, lysis was also alleviated by exhaustion of glucose from the medium which was evidenced by a lower lysis of central cells compared to peripheral ones. Thus, lysis takes place at a certain glucose concentration range that most probably provides bacteria a hunger signal. An analysis of membrane protein pattern revealed several hunger-induced changes in the bacterial outer membrane: at glucose limitation the amount of OprB1 channel protein was significantly increased whereas that of OprE was decreased. Hunger-induced up-regulation of OprB1 correlated in space and time with the lysis of the colR mutant, indicating that hunger response is detrimental to the colR-deficient bacteria. The amount of OprB1 is controlled post-transcriptionally and derepression of OprB1 in glucose-limiting medium depends at least partly on the carbon catabolite regulator protein Crc. The essentiality of ColR in hunger response can be bypassed by reducing the amount of certain outer membrane proteins. In addition to depletion of OprB1, the lysis defect of colR mutant can be suppressed by the down-regulation of OprF levels and the hindering of SecB-dependent protein secretion. CONCLUSIONS: We show that Pseudomonas putida growing on solid glucose medium adapts to glucose limitation through up-regulation of the sugar channel protein OprB1 that probably allows enhanced acquisition of a limiting nutrient. However, to survive such hunger response bacteria need signalling by the ColRS system. Hence, the ColRS system should be considered a safety factor in hunger response that ensures the welfare of the cell membrane during the increased expression of certain membrane proteins.