Isolation and characterization of a phage collection against Pseudomonas putida.

The environmental bacterium, Pseudomonas putida, possesses a broad spectrum of metabolic pathways. This makes it highly promising for use in biotechnological production as a cell factory, as well as in bioremediation strategies to degrade various aromatic pollutants. For P. putida to flourish in its environment, it must withstand the continuous threats posed by bacteriophages. Interestingly, until now, only a handful of phages have been isolated for the commonly used laboratory strain, P. putida KT2440, and no phage defence mechanisms have been characterized. In this study, we present a new Collection of Environmental P. putida Phages from Estonia, or CEPEST. This collection comprises 67 double-stranded DNA phages, which belong to 22 phage species and 9 phage genera. Our findings reveal that most phages in the CEPEST collection are more infectious at lower temperatures, have a narrow host range, and require an intact lipopolysaccharide for P. putida infection. Furthermore, we show that cryptic prophages present in the P. putida chromosome provide strong protection against the infection of many phages. However, the chromosomal toxin-antitoxin systems do not play a role in the phage defence of P. putida. This research provides valuable insights into the interactions between P. putida and bacteriophages, which could have significant implications for biotechnological and environmental applications.

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.

Direct activation of a bacterial innate immune system by a viral capsid protein.

Bacteria have evolved diverse immunity mechanisms to protect themselves against the constant onslaught of bacteriophages1-3. Similar to how eukaryotic innate immune systems sense foreign invaders through pathogen-associated molecular patterns4 (PAMPs), many bacterial immune systems that respond to bacteriophage infection require phage-specific triggers to be activated. However, the identities of such triggers and the sensing mechanisms remain largely unknown. Here we identify and investigate the anti-phage function of CapRelSJ46, a fused toxin-antitoxin system that protects Escherichia coli against diverse phages. Using genetic, biochemical and structural analyses, we demonstrate that the C-terminal domain of CapRelSJ46 regulates the toxic N-terminal region, serving as both antitoxin and phage infection sensor. Following infection by certain phages, newly synthesized major capsid protein binds directly to the C-terminal domain of CapRelSJ46 to relieve autoinhibition, enabling the toxin domain to pyrophosphorylate tRNAs, which blocks translation to restrict viral infection. Collectively, our results reveal the molecular mechanism by which a bacterial immune system directly senses a conserved, essential component of phages, suggesting a PAMP-like sensing model for toxin-antitoxin-mediated innate immunity in bacteria. We provide evidence that CapRels and their phage-encoded triggers are engaged in a 'Red Queen conflict'5, revealing a new front in the intense coevolutionary battle between phages and bacteria. Given that capsid proteins of some eukaryotic viruses are known to stimulate innate immune signalling in mammalian hosts6-10, our results reveal a deeply conserved facet of immunity.

Nonhydrolysable Analogues of (p)ppGpp and (p)ppApp Alarmone Nucleotides as Novel Molecular Tools.

While alarmone nucleotides guanosine-3',5'-bisdiphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3',5'-bisdiphosphate) and pppApp (adenosine-5'-triphosphate-3'-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the RelA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNuNpp analogues starting from 3'-azido-3'-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppGNpp as a molecular tool, we show that (i) as an HD substrate mimic, ppGNpp competes with ppGpp to inhibit the enzymatic activity of human MESH1 Small Alarmone Hyrolase, SAH; and (ii) mimicking the allosteric effects of (p)ppGpp, (p)ppGNpp acts as a positive regulator of the synthetase activity of long ribosome-associated RSHs Rel and RelA. Finally, by solving the structure of the N-terminal domain region (NTD) of T. thermophilus Rel complexed with pppGNpp, we show that as an HD substrate mimic, the analogue serves as a bona fide orthosteric regulator that promotes the same intra-NTD structural rearrangements as the native substrate.

(p)ppGpp controls stringent factors by exploiting antagonistic allosteric coupling between catalytic domains.

Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product-the alarmone nucleotide (p)ppGpp-through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity.

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.

Chromosomal toxin-antitoxin systems in Pseudomonas putida are rather selfish than beneficial.

Chromosomal toxin-antitoxin (TA) systems are widespread genetic elements among bacteria, yet, despite extensive studies in the last decade, their biological importance remains ambivalent. The ability of TA-encoded toxins to affect stress tolerance when overexpressed supports the hypothesis of TA systems being associated with stress adaptation. However, the deletion of TA genes has usually no effects on stress tolerance, supporting the selfish elements hypothesis. Here, we aimed to evaluate the cost and benefits of chromosomal TA systems to Pseudomonas putida. We show that multiple TA systems do not confer fitness benefits to this bacterium as deletion of 13 TA loci does not influence stress tolerance, persistence or biofilm formation. Our results instead show that TA loci are costly and decrease the competitive fitness of P. putida. Still, the cost of multiple TA systems is low and detectable in certain conditions only. Construction of antitoxin deletion strains showed that only five TA systems code for toxic proteins, while other TA loci have evolved towards reduced toxicity and encode non-toxic or moderately potent proteins. Analysis of P. putida TA systems' homologs among fully sequenced Pseudomonads suggests that the TA loci have been subjected to purifying selection and that TA systems spread among bacteria by horizontal gene transfer.

A nucleotide-switch mechanism mediates opposing catalytic activities of Rel enzymes.

Bifunctional Rel stringent factors, the most abundant class of RelA/SpoT homologs, are ribosome-associated enzymes that transfer a pyrophosphate from ATP onto the 3' of guanosine tri-/diphosphate (GTP/GDP) to synthesize the bacterial alarmone (p)ppGpp, and also catalyze the 3' pyrophosphate hydrolysis to degrade it. The regulation of the opposing activities of Rel enzymes is a complex allosteric mechanism that remains an active research topic despite decades of research. We show that a guanine-nucleotide-switch mechanism controls catalysis by Thermus thermophilus Rel (RelTt). The binding of GDP/ATP opens the N-terminal catalytic domains (NTD) of RelTt (RelTtNTD) by stretching apart the two catalytic domains. This activates the synthetase domain and allosterically blocks hydrolysis. Conversely, binding of ppGpp to the hydrolase domain closes the NTD, burying the synthetase active site and precluding the binding of synthesis precursors. This allosteric mechanism is an activity switch that safeguards against futile cycles of alarmone synthesis and degradation.

The Rel stringent factor from Thermus thermophilus: crystallization and X-ray analysis.

The stringent response, controlled by (p)ppGpp, enables bacteria to trigger a strong phenotypic resetting that is crucial to cope with adverse environmental changes and is required for stress survival and virulence. In the bacterial cell, (p)ppGpp levels are regulated by the concerted opposing activities of RSH (RelA/SpoT homologue) enzymes that can transfer a pyrophosphate group of ATP to the 3' position of GDP (or GTP) or remove the 3' pyrophosphate moiety from (p)ppGpp. Bifunctional Rel enzymes are notoriously difficult to crystallize owing to poor stability and a propensity for aggregation, usually leading to a loss of biological activity after purification. Here, the production, biochemical analysis and crystallization of the bifunctional catalytic region of the Rel stringent factor from Thermus thermophilus (RelTtNTD) in the resting state and bound to nucleotides are described. RelTt and RelTtNTD are monomers in solution that are stabilized by the binding of Mn2+ and mellitic acid. RelTtNTD crystallizes in space group P4122, with unit-cell parameters a = b = 88.4, c = 182.7 Å, at 4°C and in space group P41212, with unit-cell parameters a = b = 105.7, c = 241.4 Å, at 20°C.

The TonBm-PocAB System Is Required for Maintenance of Membrane Integrity and Polar Position of Flagella in Pseudomonas putida.

TonB-ExbB-ExbD-like energy transduction systems are widespread among Gram-negative bacteria. While most species have only one copy of tonB-exbBD genes, the Pseudomonas species possess more TonB-ExbBD homologues. One of them, the TonB3-PocA-PocB complex, was recently shown to be required for polar localization of FlhF and, thus, the flagella in Pseudomonas aeruginosa Here, we show that the orthologous TonBm-PocA-PocB complex is important for polar localization of FlhF and flagella in Pseudomonas putida as well. Additionally, the system is necessary for maintaining membrane integrity, as the inactivation of the TonBm-PocAB complex results in increased membrane permeability, lowered stress tolerance, and conditional cell lysis. Interestingly, the functionality of TonBm-PocAB complex is more important for stationary than for exponentially growing bacteria. The whole-cell proteome analysis provided a likely explanation for this growth phase dependence, as extensive reprogramming was disclosed in an exponentially growing tonBm deletion strain, while only a few proteomic changes, mostly downregulation of outer membrane proteins, were determined in the stationary-phase ΔtonBm strain. We propose that this response in exponential phase, involving, inter alia, activation of AlgU and ColR regulons, can compensate for TonBm-PocAB's deficiency, while stationary-phase cells are unable to alleviate the lack of TonBm-PocAB. Our results suggest that mislocalization of flagella does not cause the membrane integrity problems; rather, the impaired membrane intactness of the TonBm-PocAB-deficient strain could be the reason for the random placement of flagella.IMPORTANCE The ubiquitous Pseudomonas species are well adapted to survive in a wide variety of environments. Their success relies on their versatile metabolic, signaling, and transport ability but also on their high intrinsic tolerance to various stress factors. This is why the study of the stress-surviving mechanisms of Pseudomonas species is of utmost importance. The stress tolerance of Pseudomonads is mainly achieved through the high barrier property of their membranes. Here, we present evidence that the TonB-ExbBD-like TonBm-PocAB system is involved in maintaining the membrane homeostasis of Pseudomonas putida, and its deficiency leads to lowered stress tolerance and conditional cell lysis.

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.

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.

Desperate times call for desperate measures: benefits and costs of toxin-antitoxin systems.

Toxin-antitoxin (TA) loci were first described as killing systems for plasmid maintenance. The surprisingly abundant presence of TA loci in bacterial chromosomes has stimulated an extensive research in the recent decade aimed to understand the biological importance of these potentially deadly systems. Accumulating evidence suggests that the evolutionary success of genomic TA systems could be explained by their ability to increase bacterial fitness under stress conditions. While TA systems remain quiescent under favorable growth conditions, the toxins can be activated in response to stress resulting in growth suppression and development of stress-tolerant dormant state. Yet, several studies suggest that the TA-mediated stress protection is costly and traded off against decreased fitness under normal growth conditions. Here, we give an overview of the fitness benefits of the chromosomal TA systems, and discuss the costs of TA-mediated stress protection.

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.