Добірка наукової літератури з теми "Bacterial Toxin-antitoxin"

ABSTRACT Toxin-antitoxin (TA) systems are ubiquitous in bacteria and archaea, where they play a pivotal role in the establishment and maintenance of dormancy. Under normal growth conditions, the antitoxin neutralizes the toxin. However, under conditions of stress, such as nutrient starvation or antibiotic treatment, cellular proteases degrade the antitoxin, and the toxin functions to arrest bacterial growth. We characterized the specificity determinants of the interactions between VapB antitoxins and VapC toxins from nontypeable Haemophilus influenzae (NTHi) in an effort to gain a better understanding of how antitoxins control toxin activity and bacterial persistence. We studied truncated and full-length antitoxins with single amino acid mutations in the toxin-binding domain. Coexpressing the toxin and antitoxin in Escherichia coli and measuring bacterial growth by dilution plating assayed the ability of the mutant antitoxins to neutralize the toxin. Our results identified two single amino acid residues (W48 and F52) in the C-terminal region of the VapB2 antitoxin necessary for its ability to neutralize its cognate VapC2 toxin. Additionally, we attempted to alter the specificity of VapB1 by making a mutation that would allow it to neutralize its noncognate toxin. A mutation in VapB1 to contain the tryptophan residue identified herein as important in the VapB2-VapC2 interaction resulted in a VapB1 mutant (the T47W mutant) that binds to and neutralizes both its cognate VapC1 and noncognate VapC2 toxins. This represents the first example of a single mutation causing relaxed specificity in a type II antitoxin. IMPORTANCE Toxin-antitoxin systems are of particular concern in pathogenic organisms, such as nontypeable Haemophilus influenzae , as they can elicit dormancy and persistence, leading to chronic infections and failure of antibiotic treatment. Despite the importance of the TA interaction, the specificity determinants for VapB-VapC complex formation remain uncharacterized. Thus, our understanding of how antitoxins control toxin-induced dormancy and bacterial persistence requires thorough investigation of antitoxin specificity for its cognate toxin. This study characterizes the crucial residues of the VapB2 antitoxin from NTHi necessary for its interaction with VapC2 and provides the first example of a single amino acid change altering the toxin specificity of an antitoxin.

Toxin–antitoxin (TA) systems interfere with essential cellular processes and are implicated in bacterial lifestyle adaptations such as persistence and the biofilm formation. Here, we present structural, biochemical, and functional data on an uncharacterized TA system, the COG5654–COG5642 pair. Bioinformatic analysis showed that this TA pair is found in 2,942 of the 16,286 distinct bacterial species in the RefSeq database. We solved a structure of the toxin bound to a fragment of the antitoxin to 1.50 Å. This structure suggested that the toxin is a mono-ADP-ribosyltransferase (mART). The toxin specifically modifies phosphoribosyl pyrophosphate synthetase (Prs), an essential enzyme in nucleotide biosynthesis conserved in all organisms. We propose renaming the toxin ParT for Prs ADP-ribosylating toxin and ParS for the cognate antitoxin. ParT is a unique example of an intracellular protein mART in bacteria and is the smallest known mART. This work demonstrates that TA systems can induce bacteriostasis through interference with nucleotide biosynthesis.

Bacterial type I toxin-antitoxin systems are two-component genetic modules that encode a stable toxic protein whose ectopic overexpression can lead to growth arrest or cell death, and an unstable RNA antitoxin that inhibits toxin translation during growth. These systems are widely spread among bacterial species. Type I antitoxins are cis- or trans-encoded antisense small RNAs that interact with toxin-encoding mRNAs by pairing, thereby inhibiting toxin mRNA translation and/or inducing its degradation. Under environmental stress conditions, the up-regulation of the toxin and/or the antitoxin degradation by specific RNases promote toxin translation. Most type I toxins are small hydrophobic peptides with a predicted α-helical transmembrane domain that induces membrane depolarization and/or permeabilization followed by a decrease of intracellular ATP, leading to plasmid maintenance, growth adaptation to environmental stresses, or persister cell formation. In this review, we describe the current state of the art on the folding and the membrane interactions of these membrane-associated type I toxins from either Gram-negative or Gram-positive bacteria and establish a chronology of their toxic effects on the bacterial cell. This review also includes novel structural results obtained by NMR concerning the sprG1-encoded membrane peptides that belong to the sprG1/SprF1 type I TA system expressed in Staphylococcus aureus and discusses the putative membrane interactions allowing the lysis of competing bacteria and host cells.

Abstract Toxin–antitoxin (TA) systems are proposed to play crucial roles in bacterial growth under stress conditions such as phage infection. The type III TA systems consist of a protein toxin whose activity is inhibited by a noncoding RNA antitoxin. The toxin is an endoribonuclease, while the antitoxin consists of multiple repeats of RNA. The toxin assembles with the individual antitoxin repeats into a cyclic complex in which the antitoxin forms a pseudoknot structure. While structure and functions of some type III TA systems are characterized, the complex assembly process is not well understood. Using bioinformatics analysis, we have identified type III TA systems belonging to the ToxIN family across different Escherichia coli strains and found them to be clustered into at least five distinct clusters. Furthermore, we report a 2.097 Å resolution crystal structure of the first E. coli ToxIN complex that revealed the overall assembly of the protein-RNA complex. Isothermal titration calorimetry experiments showed that toxin forms a high-affinity complex with antitoxin RNA resulting from two independent (5′ and 3′ sides of RNA) RNA binding sites on the protein. These results further our understanding of the assembly of type III TA complexes in bacteria.

Most prokaryotic chromosomes contain a number of toxin–antitoxin (TA) modules consisting of a pair of genes that encode 2 components, a stable toxin and its cognate labile antitoxin. TA systems are also known as addiction modules, since the cells become “addicted” to the short-lived antitoxin product (the unstable antitoxin is degraded faster than the more stable toxin) because its de novo synthesis is essential for their survival. While toxins are always proteins, antitoxins are either RNAs (type I, type III) or proteins (type II). Type II TA systems are widely distributed throughout the chromosomes of almost all free-living bacteria and archaea. The vast majority of type II toxins are mRNA-specific endonucleases arresting cell growth through the mechanism of RNA cleavage, thus preventing the translation process. The physiological role of chromosomal type II TA systems still remains the subject of debate. This review describes the currently known type II toxins and their characteristics. The different hypotheses that have been proposed to explain their role in bacterial physiology are also discussed.

Toxin–antitoxin (TA) systems, which are ubiquitously present in plasmids, bacterial and archaeal genomes, are classified as types I to VI, according to the nature of the antitoxin and to the mode of toxin inhibition [...]

Bacterial persistence is considered as one of the primary reason for antibiotic tolerance besides genetically acquired antibiotic resistance. Persisters are the subpopulation of a clonal bacterial population, which can survive environmental extremes and become invulnerable to stresses due to limited metabolic activities and physiological functions. Cognate toxin and antitoxin (TA) pairs, which are transcribed simultaneously from the same or different operons within the bacterial chromosomes or plasmids, play an important role for bacterial survival during stressful growth environments. Pseudomonas aeruginosa PAO1 is one of the most versatile microorganisms in the environment. Despite its ubiquitous presence, no studies have shown the differential expression pattern of its toxin-antitoxins, and persistence related genes. The purpose of the following study is to analyze differential expression of P. aeruginosa PAO1 type II toxin-antitoxins and persistence related genes under different growth conditions and to show how their stoichiometric ratio changes during different growth conditions. Differential expression analysis indicated that the toxins and antitoxin pairs behave differently under different growth conditions. In addition, the genes related to persistence presented relatively consistent differential expression pattern under different growth environment.
Master of Science

All bacteria initiate translation using formylated methionine, yet directly after translation, the formyl-group is removed. This sequence of addition and removal appears futile, yet every sequenced bacterial genome encodes the enzymes for formylation and deformylation, suggesting this process is essential. Puzzlingly, the process is absent from both Archaea and Eukaryotes, and moreover, bacterial mutants lacking both the formylase and deformylase activities are viable, albeit with a diminished growth rate. We created an Escherichia coli strain devoid of formylase and deformylase activity. This strain was then allowed to evolve over 1500 generations whereupon it reached wild-type growth rate, demonstrating that formylation can be completely dispensed with. This raises an additional question: if the formylation cycle is unnecessary, how did it emerge and why has it persisted? Our results show that the formylation-deformylation cycle could have evolved as a toxin-antitoxin pair (TA) with post-segregational killing (PSK) activity. TAs ‘addict’ cells to the plasmids that carry them by inducing PSK. We measured the stability of formylase-deformylase encoding plasmids and their ability to elicit PSK in our evolved E. coli strain. We report several lines of evidence consistent with the formylation-cycle having evolved from a plasmid-borne PSK element: 1) in the absence of deformylation, formyl-methionine on proteins is cytotoxic in bacteria 2) deformylation relieves the cytotoxicity of formyl-methionine, 3) the loss of a plasmid containing formylase and deformylase genes from evolved cells results in cessation of growth – a standard PSK phenotype. In addition, we introduced the E. coli formylase and deformylase genes into yeast and demonstrate that Met-tRNA formylation is not lethal, even in the absence of deformylation. This suggests PSK would be ineffectual in yeast, accounting for the absence of formylation from eukaryotic cytoplasmic translation. We also report the presence of formylase and deformylase genes in the two representative members of the archaeal Methanocopusculum genus. Moreover, we demonstrate that these genes have been acquired by a recent horizontal gene transfer from bacteria. Our results indicate that formylmethionine use in bacteria evolved, not through a direct functional benefit to cells, but through competition between infectious genetic elements.

Les systèmes toxine-antitoxine (TA) bactériens ont été découverts il y a une vingtaine d’année sur les plasmides à bas nombre de copie. Ils sont composés de deux gènes organisés en opéron, l’un codant pour une toxine stable et l’autre pour une antitoxine instable capable de neutraliser l’effet de la toxine. Les systèmes TA sont fortement représentés au sein de l’ensemble des génomes bactériens. Ils se localisent aussi bien sur des éléments génétiques mobiles (plasmides, phages, transposons,…) que dans les chromosomes, ce qui suggère que le transfert horizontal de gènes participe à leur dissémination. Le système TA ccd du plasmide F d’Escherichia coli (ccdF) est composé de l’antitoxine CcdA et de la toxine CcdB. Le système ccdF contribue à la stabilité du plasmide F en tuant les bactéries-filles n’ayant pas reçu de copies plasmidiques lors de la division bactérienne (tuerie post-ségrégationelle).

Au cours de ce travail, nous avons caractérisé un homologue du système toxine-antitoxine ccd du plasmide F (ccdF) qui se situe dans le chromosome de la souche pathogène E. coli O157:H7 EDL933 entre les gènes folA et apaH (ccdO157). Les systèmes ccdF et ccdO157 coexistent naturellement dans les souches d’E. coli O157:H7, le système ccdF se trouvant sur le plasmide pO157 qui dérive du plasmide F. Nos résultats montrent que l’antitoxine plasmidique CcdAF neutralise l’effet de la toxine chromosomique CcdBO157, tandis que l’antitoxine chromosomique CcdAO157 ne contrecarre pas la toxicité de la toxine plasmidique CcdBF. Nous avons également montré que le système ccdF cause une tuerie post-ségrégationelle, lorsqu’il est cloné dans un plasmide instable, dans une souche possédant le système chromosomique ccdO157. Le système ccdF est donc fonctionnel en présence de son homologue chromosomique.

Le système ccdO157 est absent du chromosome de la souche de laboratoire E. coli K-12 MG1655, où une région intergénique de 77 pb sépare les gènes folA et apaH. Celle-ci contient une séquence cible pour la transposition. Nous avons étudié la distribution du système ccdO157 au sein de 523 souches d’E. coli représentatives de l’ensemble des sérogroupes décrits. Nos résultats montrent que le système ccdO157 est présent au sein de souches appartenant à 47 sérogroupes différents. Nos résultats mettent en évidence la diversité de la région intergénique folA-apaH d’E. coli. Celle-ci peut contenir gènes codant pour des protéines présentant de l’homologie avec des protéines d’espèce bactériennes éloignées d’E. coli ou d’organismes eucaryotes, ainsi qu’un élément génétique mobile, l’IS621, ce qui montre que le système ccdO157 a intégré le chromosome d’E. coli via le transfert horizontal de gènes.

Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2015.
Title as it appears in MIT Commencement Exercises program, June, 2015: Toxin-antitoxin systems in bacteria : targets, mechanisms, and specificity. Cataloged from PDF version of thesis.
Includes bibliographical references.
Toxin-antitoxin (TA) systems are genetic modules widely present on bacterial chromosomes. These systems comprise a toxin and cognate antitoxin that are encoded together in an operon; normally, the toxin and antitoxin are synthesized and form a non-toxic complex. Under times of stress, however, the more labile antitoxin can be degraded, which frees the toxin to inhibit growth. TA systems have been implicated in a number of important processes, including plasmid stability, phage resistance, persistence, and virulence. Yet, there are a number of unanswered questions about these genetic modules. What are the cellular targets of toxins? How do antitoxins antagonize their cognate toxins? Do toxins and antitoxins interact in a one-to-one manner - one antitoxin for one toxin - or do they form large networks of cross-reacting systems? To answer these questions, I have studied the targets, mechanisms, and specificity of TA systems in bacteria. For my first project, I identified SocAB, a novel TA system in the bacterium Caulobacter crescentus. Unlike canonical TA systems, in which the antitoxin is less stable than the toxin, I found that the toxin SocB is unstable and constitutively degraded by the protease ClpXP. This degradation requires its antitoxin, SocA, which acts a proteolytic adaptor. Furthermore, I found that SocB blocks replication progress through an interaction with the sliding clamp, thus expanding the number of known cellular targets for TA systems. For my second project, I studied interaction specificity in the ParDE TA family. I found that toxins and antitoxins in this family exhibit a strong preference for interacting with their cognate pair, and that specificity is determined by a small subset of coevolving residues at the interface of these two proteins. To understand how the identity of these coevolving residues controls interaction specificity, I generated a library of ~10⁴ variants at these coevolving positions in the ParD antitoxin. By reacting this library against both cognate and non-cognate ParE toxins, I identified promiscuous ParD variants that are densely connected to specific variants in sequence space. These promiscuous states may facilitate changes in TA specificity and promote the expansion of these paralogous systems by duplication and divergence.
by Christopher David Aakre.
Ph. D.

In the last few decades, bacterial Toxin-antitoxin (TA) systems have been identified to play crucial roles in bacterial survival under stressful conditions and virulence. TA systems are pair of genetic elements where one of the genes codes for a protein (toxin), which is toxic to the host cell, and the other gene code for its antidote (antitoxin), which can be an RNA or a protein. Under favourable growth conditions, the antitoxin inhibits the toxin activity; however, when the bacterial cell encounters stressful conditions such as antibiotic exposure, starvation, phage infection, etc., the toxin is released from antitoxin inhibition resulting in cell growth arrest or cell death. The TA systems have been mainly implicated in plasmid maintenance, inhibition of bacteriophage propagation (abortive infection), and survival against antibiotic exposure (persister cell formation). The current thesis work is focused on understanding the structural basis of toxin inhibition and autoregulation of operon expression in the HigBA type II TA system from E. coli. This study reports a high-resolution 2.09 Å crystal structure of the HigBA complex from E. coli K-12. This structure reveals the overall organization and mechanism of antitoxin HigA binding to toxin HigB. We also report a 2.3 Å resolution crystal structure of a truncated heterodimeric HigBA complex. This structure signifies the role of helices 𝛼1 and 𝛼2 in the dimerization of HigA. Also, we propose that the dimeric structure may indicate the possible proteolytic cleavage sites in toxin HigB and antitoxin HigA, which may have implications in HigBA complex disassembly and regulation in bacteria under proteolytic stress. Further using CD spectroscopy, NMR spectroscopy, and MD simulation studies, we suggest that E. coli HigA antitoxin is well-folded and stable in solution; however, it shows an intrinsic dynamic behavior. Using EMSA, SEC-MALS, and ITC experiments we establish that HigBA binds to its 33bp promoter DNA (Pal-1 DNA) in a 2:1 (HigA: Pal-1) ratio, and through ITC experiments we report that both the HigBA complex and HigA have comparable high binding affinity towards 33bp Pal-1 DNA. Therefore, we suggest that the toxin HigB has little or no effect on the antitoxin’s DNA binding activity. Further, the C-terminal DBD of HigA (HigA_DBD) was cloned and purified for the NMR-based titration experiments to identify the DNA binding residues. The sequential backbone assignments of HigA_DBD were achieved and using NMR CSP data from the titration experiments with different Pal-1 DNA sequences, we reveal that residues from helix 𝛼7, 𝛼8, loop L1 and loop L2 of the DNA binding domain of HigA interact with Pal-1 promoter DNA sequence. Further, we report the NMR CSP data-driven HADDOCK model of Pal-1 DNA bound HigBA complex. Finally, we report a low-resolution cryoEM structure of the HigBA and 27bp Pal-1 DNA complexes, confirming that two HigBA complexes bind the Pal-1 DNA sequence.

Bacteria adopt several defense strategies to enable their survival against the environmental threats they encounter from time to time. Toxin-antitoxin (TA) systems are being understood as a key bacterial defense mechanism against invading viruses, antibiotics, and other environmental stress. TA systems consist of a pair of genes, usually under a common promoter, that code for a toxin and its cognate antitoxin . The toxin is usually a protein, that arrests cellular growth during stress, whereas the antitoxin can be a protein or a non-coding RNA, that inhibits the toxin. The TA systems are classified into six different types based on the mechanism of inhibition of toxin by antitoxin. In type III TA systems, the toxin is an endoribonuclease (RNase) that cleaves cellular RNAs when free, whereas antitoxin is a non-coding RNA. The toxin also processes its own precursor antitoxin RNA into smaller repeats and subsequently assembles with them to form an inactive TA complex. During normal growth conditions, the antitoxin RNA inhibits the toxin protein by forming the RNA-protein TA complex. However, when the bacteria encounter stress such as phage infection, the active toxin gets released from the complex and prevents phage replication. Type III TA systems have been identified in several bacteria and classified into three different families - toxIN, cptIN, and tenpIN. However, type III systems have not been identified and well characterized in Escherichia coli. The identification and characterization of these systems in E. coli, which is the most commonly studied model organism with robust genetic manipulation tools available, would help in understanding them in detail for their functions and mechanism of action. In this thesis, by using protein sequence-based homology searches, we report the identification of ToxIN type III TA systems from several strains in E. coli. Multiple sequence alignment of the toxin protein sequences revealed that these systems could be further grouped in five different clusters and there are several conserved residue positions that could be vital for the toxin structure and function. Secondary structure analysis of representative sequences of antitoxin RNA repeats from five different clusters suggested that these RNAs have the propensity to form pseudoknot structure. Toxin-antitoxin functional assays performed using one of the identified TA systems from E. coli (strain 680) showed that the identified system indeed functions as a type III TA system. Though type III TA systems are known to be found in several organisms, very few of them have been characterized structurally and biophysically. This is mainly due to the challenges in cloning the toxin proteins in expression vectors and the lack of standard protocols to express and purify the type III TA components. Hence, we decided to establish protocols for cloning and purification of type III TA components. Here, we report the large-scale expression and purification of the toxin, antitoxin and complex components from four different type III TA systems (three from toxIN and one from tenpIN families) in E. coli. This strategy involves cloning the toxin and antitoxin coding DNA sequences in two different co-expression compatible, commercially available expression vectors. Co-transformation and co-expression of toxin and antitoxin genes in laboratory strains of E. coli led to the expression and purification of type III TA complex. Using anion exchange chromatography, we could obtain separate fractions of toxin protein, antitoxin RNA, and complex components in significant quantities suitable for biophysical experiments. Further, we were able to crystallize the type III TA complex from E. coli (strain 680) and solve the X-ray crystal structure at a resolution of 2.097 Å. This is the first reported structure of a type III TA complex from E. coli. The E. coli type III toxin and antitoxin were arranged in a cyclic heterohexameric assembly in the complex structure. This assembly was also verified in solution using SEC-MALS analysis. The toxin protein, which is an endoribonuclease, adopts a β-sheet containing core structure surrounded by α-alpha helices. The antitoxin RNA forms a pseudoknot structure with two stems and two loops and the 5′ and 3′ single-stranded regions interact with the toxin protein. The structure also uncovered the presence of several key interactions between the toxin and antitoxin and provided molecular basis for the substrate sequence specificity of the toxin. Mapping the amino acid residues which were conserved in all five clusters of E. coli toxIN, onto the structure of the E. coli type III TA complex showed that most of these residues were crucial for toxin folding and endoribonuclease activity. The multiple sequence alignment of antitoxin RNA sequences revealed that the core pseudoknot region was conserved for both sequence and structure across the five different clusters and the 5′ and 3′ single-stranded overhangs were variable, that could lead to specificity of the antitoxins to their cognate toxins. The assembly of the type III TA complex has not been studied so far in terms of toxin-antitoxin binding affinity and free energy change of the complex formation. Hence, we characterized the binding of toxin protein and antitoxin RNA using isothermal titration calorimetry (ITC) experiments. The ITC experiments reveled that the toxin and antitoxin interact with each other with a very high binding affinity in a two-step binding event. The structure of the complex showed that toxin and antitoxin possess two non-identical binding sites for each other which leads to a two-step binding process. Using truncated antitoxin RNA mutants, we could simplify the two-step binding into two one-step binding events and estimate the binding contribution from each individual site. Based on our ITC experiments on the full-length antitoxin repeat and the truncated repeats, we have proposed a model for the assembly of toxin and antitoxin into a cyclic heterohexameric complex. Using nuclear magnetic resonance (NMR) spectroscopy, we characterized the structure of the free antitoxin RNA repeat for its foldedness. The 1D 1H and the 2D 1H-1H NOESY NMR spectra showed that the free antitoxin RNA adopts a folded structure in solution. This was further confirmed by recording a 2D 1H-15N HSQC spectrum of the free antitoxin. The NMR spectra of only the core pseudoknot forming region of the antitoxin suggested that the antitoxin RNA could fold into a pseudoknot structure even in the absence of toxin protein. Perturbation of a noncanonical U-U base pair, which is part of a U:U:G triplet, in the antitoxin RNA significantly altered its structure indicating that noncanonical and tertiary interactions are crucial for antitoxin folding. The thesis has been organized as follows: Chapter-1 provides a brief review on toxin-antitoxin (TA) systems and their classification with a special focus on type III TA systems, that have been studied in this work. Chapter-2 describes the identification and functional characterization of type III TA systems in Escherichia coli. Chapter-3 details the expression and purification of toxin, antitoxin, and complex components from four different type III TA systems in E. coli. In this chapter, we also propose a standard protocol for cloning, expression, and purification of type III TA components for biophysical experiments. In Chapter-4, we report the structure of the first type III TA complex in E. coli. Our studies on toxin and antitoxin binding using isothermal titration calorimetry (ITC) are also described in this chapter. Chapter-5 details the characterization of free antitoxin RNA by solution NMR spectroscopy.

Christiaan Eijkman shared a 1929 Nobel Prize “for his discovery of the antineuritic vitamin.” His extensive studies on chickens and prison inmates on the island of Java in the 1890s helped establish a white rice diet as a cause of beriberi, and the rice coating as a remedy. Eijkman reported that he had traced a bacterial disease, its toxin, and its antitoxin. Beriberi, however, is a nutrient deficiency. Eijkman was wrong. Ironically, Eijkman even rejected the current explanation when it was first introduced in 1910. Although he earned a Nobel Prize for his important contribution on the role of diet, Eijkman’s original conclusion about the bacterium was just plain mistaken. Eijkman’s error may seem amusing, puzzling, or even downright disturbing—an exception to conventional expectations. Isn’t the scientific method, properly applied, supposed to protect science from error? And who can better exemplify science than Nobel Prize winners? If not, how can we trust science? And who else is to serve as role models for students and aspiring scientists? Eijkman’s case, however, is not unusual. Nobel Prize–winning scientists have frequently erred. Here I profile a handful of such cases (Figure 11.1). Among them is one striking pair, Peter Mitchell and Paul Boyer, who advocated alternative theories of energetics in the cell. Each used his perspective to understand and correct an error of the other! Ultimately, all these cases offer an occasion to reconsider another sacred bovine—that science is (or should be) free of error, and that the measure of a good scientist is how closely he or she meets that ideal. Consider first Linus Pauling, the master protein chemist. Applying his intimate knowledge of bond angles, he deciphered the alpha-helix structure of proteins in 1950, which earned him a Nobel Prize in 1954. He also reasoned fruitfully about sickle cell hemoglobin, leading to molecular understanding of its altered protein structure. Yet Pauling also believed that megadoses of vitamin C could cure the common cold. Evidence continues to indicate otherwise, although Pauling’s legacy still seems to shape popular beliefs.