Ditlev Brodersen

Aarhus University | AU · Department of Molecular Biology and Genetics

Phosphorus is an essential macronutrient for all microorganisms and can be extracted from the environment by several metabolic pathways. In Escherichia coli, the 14-cistron phn operon encoding the carbon-phosphorus (C-P) lyase enzymatic machinery allows for extraction of phosphorus from a wide range of phosphonates characterised by the highly stabl...

Many bacteria encode multiple toxin-antitoxin (TA) systems targeting separate, but closely related, cellular functions. The toxin of the E. coli hipBA system, HipA, is a kinase that inhibits inhibits translation via phosphorylation of glutamyl-tRNA synthetase. Enteropathogenic E. coli (EPEC) O127:H6 encodes an additional, tripartite TA module, hipB...

Bacterial multidrug tolerance and persistence are problems of increasing scientific and medical significance. The first gene discovered to confer persistence was hipAhipBAE. coli

Toxin – Antitoxin modules function in the genetic stability of mobile genetic elements, bacteriophage defense, and antibiotic tolerance. A gain-of-function mutation of the Escherichia coli K-12 hipBA module can induce antibiotic tolerance in a subpopulation of bacterial cells, a phenomenon known as persistence. HipA is a Ser/Thr kinase that phospho...

Bacterial type II toxin-antitoxin (TA) modules encode a toxic protein that downregulates metabolism and a specific antitoxin that binds and inhibits the toxin during normal growth. In non-typeable Haemophilus influenzae, a common cause of infections in humans, the vapXD locus was found to constitute a functional TA module and contribute to pathogen...

A hallmark of type I CRISPR–Cas systems is the presence of Cas3, which contains both the nuclease and helicase activities required for DNA cleavage during interference. In subtype I-D systems, however, the histidine-aspartate (HD) nuclease domain is encoded as part of a Cas10-like large effector complex subunit and the helicase activity in a separa...

Phenomycin is a bacterial mini-protein of 89 amino acids discovered more than 50 years ago with toxicity in the nanomolar regime toward mammalian cells. The protein inhibits the function of the eukaryotic ribosome in cell-free systems and appears to target translation initiation. Several fundamental questions concerning the cellular activity of phe...

Phenomycin is a bacterial mini-protein of 89 amino acids discovered more than 50 years ago with toxicity in the nanomolar regime towards mammalian cells. The protein inhibits the function of the eukaryotic ribosome in cell free systems and appears to target translation initiation. Several fundamental questions concerning the cellular activity of ph...

In our recent publication (Zhang et al., 2019), we demonstrate an interesting mode of regulation of purine metabolism unique to Proteobacteria. In this microreview, we would like to reflect on the ideas put forward, with special focus on protein domain architecture of the enzyme involved, its orthologues in plants, and the implications of the diffe...

The E. coli hicAB type II toxin-antitoxin locus is unusual by being controlled by two promoters and by having the toxin encoded upstream of the antitoxin. HicA toxins contain a double-stranded RNA-binding fold and cleaves both mRNA and tmRNA in vivo, while HicB antitoxins contain a partial RNase H fold and either a helix-turn-helix (HTH) or ribbon-...

Bacterial toxin-antitoxin (TA) modules confer multidrug tolerance (persistence) that may contribute to the recalcitrance of chronic and recurrent infections. The first high-persister gene identified was hipA of Escherichia coli strain K-12, which encodes a kinase that inhibits glutamyl-tRNA synthetase. The hipA gene encodes the toxin of the hipBA T...

The stringent response alarmones pppGpp and ppGpp are essential for rapid adaption of bacterial physiology to changes in the environment. In Escherichia coli, the nucleosidase PpnN (YgdH) regulates purine homeostasis by cleaving nucleoside monophosphates and specifically binds (p)ppGpp. Here, we show that (p)ppGpp stimulates the catalytic activity...

Ribosyl 1,5-bisphosphate (PRibP) was discovered 65 years ago and was believed to be an important intermediate in ribonucleotide metabolism, a role immediately taken over by its "big brother" phosphoribosyldiphosphate. Only recently has PRibP come back into focus as an important player in the metabolism of ribonucleotides with the discovery of the p...

Type II toxin‐antitoxin (TA) modules, which are important cellular regulators in prokaryotes, usually encode two proteins, a toxin that inhibits cell growth and a non‐toxic and labile inhibitor (antitoxin) that binds to and neutralizes the toxin. Here, we demonstrate that the res‐xre locus from Photorhabdus luminescens and other bacterial species f...

Toxin‐antitoxin (TA) systems have been strongly implicated in bacterial cell survival during antibiotic stress and are likely to play important roles in persister cell formation1,2. Understanding the function and regulation of TA systems will thus be vital for combatting chronic and recurrent human infections in the future. Expression of type II TA...

Bacteria have evolved advanced strategies for surviving during nutritional stress, including expression of specialized enzyme systems that allow them to grow on unusual nutrient sources. Inorganic phosphate (Pi) is limiting in most ecosystems, hence organisms have developed a sophisticated, enzymatic machinery known as carbon‐phosphorus (C‐P) lyase...

In the original version of this Article, molecular weight markers in Figs 1c, 2c,d and 4d were displaced during the production process, so that they were not correctly aligned with the corresponding bands. In addition, in Fig. 4c, molecular masses given for three different elution volumes were displaced so that they appeared to the left of the corr...

Viruses employ a range of strategies to counteract the prokaryotic adaptive immune system, clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas), including mutational escape and physical blocking of enzymatic function using anti-CRISPR proteins (Acrs). Acrs have been found in many bacteriophages but s...

Bacterial toxin-antitoxin (TA) modules are abundant genetic elements that encode a toxin protein capable of inhibiting cell growth and an antitoxin that counteracts the toxin. The majority of toxins are enzymes that interfere with translation or DNA replication, but a wide variety of molecular activities and cellular targets have been described. An...

The stringent response is a global reprogramming of bacterial physiology that renders cells more tolerant to antibiotics and induces virulence gene expression in pathogens in response to stress. This process is driven by accumulation of the intracellular alarmone guanosine-5'-di(tri)phosphate-3'-diphosphate ([p]ppGpp), which is produced by enzymes...

The PIN domain (Pil N-terminus) is a compact RNA-binding protein domain present in all domains of life. This 120-residue domain consists of a central and parallel β sheet surrounded by α helices, which together organise 4-5 acidic residues in an active site that binds one or more divalent metal ions and in many cases has endoribonuclease activity....

Toxin-antitoxin systems are widespread in the bacterial kingdom, including in pathogenic species, where they allow rapid adaptation to changing environmental conditions through selective inhibition of key cellular processes, such as DNA replication or protein translation. Under normal growth conditions, type II toxins are inhibited through tight pr...

Expression of bacterial type II toxin-antitoxin (TA) systems is regulated at the transcriptional level through direct binding of the antitoxin to pseudo-palindromic sequences on operator DNA. In this context, the toxin functions as a co-repressor by stimulating DNA binding through direct interaction with the antitoxin. Here, we determine crystal st...

Sansalvamide is a cyclic pentadepsipeptide produced by Fusarium solani and has shown promising results as potential anti-cancer drug. The biosynthetic pathway has until now remained unidentified, but here we used an Agrobacterium tumefaciens-mediated transformation (ATMT) approach to generate knockout mutants of two candidate non-ribosomal peptide...

The Gal repressor regulates transport and metabolism of D-galactose in Escherichia coli and can mediate DNA loop formation by forming a bridge between adjacent or distant sites. GalR forms insoluble aggregates at lower salt concentrations in vitro, which can be solubilized at higher salt concentrations. Here, we investigate the assembly and disasse...

RNA decay is an important process that is essential for controlling the abundance, quality and maturation of transcripts. In eukaryotes, RNA decay in the 3′–5′ direction is carried out by the exosome, an RNA-degradation machine that is conserved from yeast to humans. A range of cofactors stimulate the enzymatic activity of the exosome and serve as...

The five module non-ribosomal peptide synthetase NRPS30 is responsible for sansalvamide biosynthesis in Fusarium solani

The VapC toxin from the Shigella flexneri 2a virulence plasmid pMYSH6000 belongs to the PIN domain protein family, which is characterized by a conserved fold with low amino acid sequence conservation. The toxin is a bona fide Mg 21-dependent ribonuclease and has been shown to target initiator tRNA fMet in vivo. Here, we present crystal structures o...

Upon infection of Escherichia coli by bacteriophage Qβ, the virus-encoded β-subunit recruits host translation elongation factors EF-Tu and EF-Ts and ribosomal protein S1 to form the Qβ replicase holoenzyme complex, which is responsible for amplifying the Qβ (+)-RNA genome. Here, we use X-ray crystallography, NMR spectroscopy, as well as sequence co...

Motivation: By using a class of large modular enzymes known as Non-Ribosomal Peptide Synthetases (NRPS), bacteria and fungi are capable of synthesizing a large variety of secondary metabolites, many of which are bioactive and have potential, pharmaceutical applications as e.g.~antibiotics. There is thus an interest in predicting the compound synth...

ECM2015

Phosphorus is required for all life and microorganisms can extract it from their environment through several metabolic pathways. When phosphate is in limited supply, some bacteria are able to use phosphonate compounds, which require specialized enzymatic machinery to break the stable carbon-phosphorus (C-P) bond. Despite its importance, the details...

Activation of toxin-antitoxin (TA) systems provides an important mechanism for bacteria to adapt to challenging and ever changing environmental conditions. Known TA systems are classified into five families based on the mechanisms of antitoxin inhibition and toxin activity. For type II TA systems, the toxin is inactivated in exponentially growing c...

Phosphorus is an essential element for all living cells and is usually taken up in the form of phosphate. A number of microorganisms, however, are capable of extracting phosphorous from organic phosphonate compounds, which are characterized by a stable carbon-phosphorus (C-P) bond (1). The metabolic pathway responsible for phosphonate degradation i...

Positive-stranded RNA viruses are common among human pathogenic viruses, which often cooperate with host proteins to fulfill essential functions during infection. One function is replication of the viral genome. The Qβ phage is a positive-stranded RNA virus that infects E.coli. The Qβ replicase holo enzyme comprises the phage-encoded RNA-dependent...

Iron is an essential, yet often limiting element for the growth of many organisms. In response to iron limitation, fungi have developed siderophores that provide a high-affinity iron uptake system and safe intracellular storage and transport mechanisms to gain a competitive advantage. Here, we discuss the function of siderophores in relation to fun...

The THO complex participates during eukaryotic mRNA biogenesis in coupling transcription to formation and nuclear export of translation-competent messenger ribonucleoprotein particles. In Saccharomyces cerevisiae, THO has been defined as a heteropentamer composed of the Tho2p, Hpr1p, Tex1p, Mft1p, and Thp2p subunits and the overall three-dimensiona...

The highly persistent and often lethal human pathogen, Mycobacterium tuberculosis contains at least 88 toxin-antitoxin genes. More than half of these encode VapC PIN domain endoribonucleases that inhibit cell growth by unknown mechanisms. Here we show that VapC20 of M. tuberculosis inhibits translation by cleavage of the Sarcin-Ricin loop (SRL) of...

Sub2p/UAP56 is a highly conserved DEAD-box RNA helicase involved in the packaging and nuclear export of mRNA/protein particles (mRNPs). In Saccharomyces cerevisiae, Sub2p is recruited to active chromatin by the pentameric THO complex and incorporated into the larger transcription-export (TREX) complex. Sub2p also plays a role in the maintenance of...

Upon release from the stable complex formed with its antitoxin VapB, the toxin VapC (MvpT) of the Gram-negative pathogen Shigella flexneri is capable of globally down-regulating translation by specifically cleaving initiator tRNA(fMet) in the anticodon region. Recombinant Shigella flexneri VapC(D7A) harbouring an active-site mutation was overexpres...

In this paper, a simple low-cost alternative to large commercial systems for preparing macromolecular crystallization conditions is described. Using an intuitive spreadsheet-based approach, the system allows the rapid calculation of relevant pipetting volumes given known stock-solution concentrations and incorporates the automatic design of custom...

The bacterial relBE locus encodes a toxin-antitoxin complex in which the toxin, RelE, is capable of cleaving mRNA in the ribosomal A site cotranslationally. The antitoxin, RelB, both binds and inhibits RelE, and regulates transcription through operator binding and conditional cooperativity controlled by RelE. Here, we present the crystal structure...

Toxin-antitoxin (TA) loci are common in archaea and prokaryotes and allow cells to rapidly adapt to changing environmental conditions through release of active regulators of metabolism. Many toxins are endonucleases that target cellular mRNA and tRNAs, while the antitoxins tightly wrap around the toxins to inhibit them under normal circumstances. T...

Deadenylation is the first and rate-limiting step during turnover of mRNAs in eukaryotes. In the yeast, Saccharomyces cerevisiae, two distinct 3′–5′ exonucleases, Pop2p and Ccr4p, have been identified within the Ccr4-NOT deadenylase complex, belonging to the DEDD and Exonuclease–Endonuclease–Phosphatase (EEP) families, respectively. Ngl3p has been...

The vertebrate 2-5A system is part of the innate immune system and central to cellular antiviral defense. Upon activation by viral double-stranded RNA, 5′-triphosphorylated, 2′–5′-linked oligoadenylate polyribonucleotides (2-5As) are synthesized by one of several 2′–5′-oligoadenylate synthetases. These unusual oligonucleotides activate RNase L, an...

Translational control is widely used to adjust gene expression levels. During the stringent response in bacteria, mRNA is degraded on the ribosome by the ribosome-dependent endonuclease, RelE. The molecular basis for recognition of the ribosome and mRNA by RelE and the mechanism of cleavage are unknown. Here, we present crystal structures of E. col...

Hfq proteins are common in many species of enterobacteria, where they participate in RNA folding and translational regulation through pairing of small RNAs and messenger RNAs. Hfq proteins share the distinctive Sm fold, and form ring-shaped structures similar to those of the Sm/Lsm proteins regulating mRNA turnover in eukaryotes. However, bacterial...

The exosome is a multi-subunit 3'-5' exonucleolytic complex that is conserved in structure and function in all eukaryotes studied to date. The complex is present in both the nucleus and cytoplasm, where it continuously works to ensure adequate quantities and quality of RNAs by facilitating normal RNA processing and turnover, as well as by participa...

In eukaryotic organisms, initiation of mRNA turnover is controlled by progressive shortening of the poly-A tail, a process involving the mega-Dalton Ccr4-Not complex and its two associated 3'-5' exonucleases, Ccr4p and Pop2p (Caf1p). RNA degradation by the 3'-5' DEDDh exonuclease, Pop2p, is governed by the classical two metal ion mechanism traditio...

Production of aberrant messenger ribonucleoprotein particles (mRNPs) is subject to quality control (QC). In yeast strains carrying mutations of the THO complex, transcription induction triggers a number of interconnected QC phenotypes: (1) rapid degradation of several mRNAs; (2) retention of a fraction of THO-dependent mRNAs in transcription site-a...

The overall fidelity of RNA biosynthesis and processing is very high. This goes for both mRNAs, which are turned over relatively quickly, and for stable RNAs, such as the components of the translational apparatus, the transfer and ribosomal RNAs. However, no enzymatic process is completely error-free, so to minimize the number of non-functional tra...

The Sm and Sm-like proteins are conserved in all three domains of life and have emerged as important players in many different RNA-processing reactions. Their proposed role is to mediate RNA-RNA and/or RNA-protein interactions. In marked contrast to eukaryotes, bacteria appear to contain only one distinct Sm-like protein belonging to the Hfq family...

Deadenylation is the first and probably also rate-limiting step of controlled mRNA decay in eukaryotes and therefore central for the overall rate of gene expression. In yeast, the process is maintained by the mega-Dalton Ccr4-Not complex, of which both the Ccr4p and Pop2p subunits are 3′–5′ exonucleases potentially responsible for the deadenylation...

The Sm and Sm-like proteins are conserved in all three domains of life. Their proposed role is to mediate RNA-RNA and/or RNA-protein interactions. Eukaryotes encode several different Sm and Sm-like proteins that form heteroheptamers with each other respectively. In contrast, bacteria appear to contain only one distinct Sm-related protein belonging...

In the paper by Brodersen et al. [(2000), Acta Cryst. D56, 431–441] errors were published in values in the last set of fractional coordinates and in the molecular mass. The correct values are given in this article.

The multisubunit eukaryotic exosome is an essential RNA processing and degradation machine. In its nuclear form, the exosome associates with the auxiliary factor Rrp6p, which participates in both RNA processing and degradation reactions. The crystal structure of Saccharomyces cerevisiae Rrp6p displays a conserved RNase D core with a flanking HRDC (...

During protein synthesis, translational release factors catalyze the release of the polypeptide chain when a stop codon on the mRNA reaches the A site of the ribosome. The detailed mechanism of this process is currently unknown. We present here the crystal structures of the ribosome from Thermus thermophilus with RF1 and RF2 bound to their cognate...

Ribosomal proteins hold a unique position in biology because their function is so closely tied to the large rRNAs of the ribosomes in all kingdoms of life. Following the determination of the complete crystal structures of both the large and small ribosomal subunits from bacteria, the functional role of the proteins has often been overlooked when fo...

The methods involved in determining the 850 kDa structure of the 30S ribosomal subunit from Thermus thermophilus were in many ways identical to those that are generally used in standard protein crystallography. This paper reviews and analyses the methods that can be used in phasing such large structures and shows that the anomalous signal collected...

Hygromycin B is an aminoglycoside antibiotic active against prokaryotic and eukaryotic ribosomes. Ribosomal alterations in bacteria conferring resistance to hygromycin B have not been described, prompting us to use a single rRNA allelic derivative of the gram-positive bacterium Mycobacterium smegmatis for investigation of the molecular mechanisms i...

Because some translation factors mimic the shape of tRNA, they were thought to bind the ribosome in a similar manner. Recent work suggests that this idea of molecular mimicry needs to be reassessed.

We present a detailed analysis of the protein structures in the 30 S ribosomal subunit from Thermus thermophilus, and their interactions with 16 S RNA based on a crystal structure at 3.05 A resolution. With 20 different polypeptide chains, the 30 S subunit adds significantly to our data base of RNA structure and protein-RNA interactions. In additio...

We describe the crystallization and structure determination of the 30 S ribosomal subunit from Thermus thermophilus. Previous reports of crystals that diffracted to 10 A resolution were used as a starting point to improve the quality of the diffraction. Eventually, ideas such as the addition of substrates or factors to eliminate conformational hete...

Crystal structures of the 30S ribosomal subunit in complex with messenger RNA and cognate transfer RNA in the A site, both in the presence and absence of the antibiotic paromomycin, have been solved at between 3.1 and 3.3 angstroms resolution. Cognate transfer RNA (tRNA) binding induces global domain movements of the 30S subunit and changes in the...

Excerpt The two subunits that make up the ribosome have bothdistinct and cooperative functions. The 30S ribosomalsubunit binds messenger RNA (mRNA) and is involvedin the selection of cognate transfer RNA (tRNA) by monitoring codon–anticodon base-pairing during the decoding process. The 50S subunit catalyzes peptide-bond formation. Both subunits wor...

Initiation of translation at the correct position on messenger RNA is essential for accurate protein synthesis. In prokaryotes, this process requires three initiation factors: IF1, IF2, and IF3. Here we report the crystal structure of a complex of IF1 and the 30S ribosomal subunit. Binding of IF1 occludes the ribosomal A site and flips out the func...

We have used the recently determined atomic structure of the 30S ribosomal subunit to determine the structures of its complexes with the antibiotics tetracycline, pactamycin, and hygromycin B. The antibiotics bind to discrete sites on the 30S subunit in a manner consistent with much but not all biochemical data. For each of these antibiotics, inter...

Genetic information encoded in messenger RNA is translated into protein by the ribosome, which is a large nucleoprotein complex comprising two subunits, denoted 30S and 50S in bacteria. Here we report the crystal structure of the 30S subunit from Thermus thermophilus, refined to 3 A resolution. The final atomic model rationalizes over four decades...

The 30S ribosomal subunit has two primary functions in protein synthesis. It discriminates against aminoacyl transfer RNAs that do not match the codon of messenger RNA, thereby ensuring accuracy in translation of the genetic message in a process called decoding. Also, it works with the 50S subunit to move the tRNAs and associated mRNA by precisely...

Two examples of the application of single-wavelength anomalous dispersion (SAD) in macromolecular structure determination are described, both using the statistical phasing program SHARP. For the holmium-substituted calcium-binding protein psoriasin (22.7 kDa), a set of accurate phases has been obtained to a resolution of 1.05 A without recourse to...

Metal ions are used in a variety of ways in our cells to regulate, activate, and stabilise specific protein molecules. In this review, we describe some of the regulatory functions of calcium and zinc that have been examined using X-ray crystallographic structural studies of specific proteins. These studies indicate that very precise control of cell...

XAct is a stand-alone application for Microsoft Windows™ 95/98 that, through a spreadsheet-like interface, allows the user to construct crystallization experiments in a highly flexible way. The object-oriented data structure helps to maintain an overview of many experiments and provides reusable stock and tray objects. The program features a hardwa...

The crystal structure of human psoriasin (S100A7) in the native, calcium-bound form has been determined from two crystal forms of the protein crystallized with and without divalent zinc. The overall structures of the dimeric protein closely resemble the previously determined holmium-substituted structure. The structures also reveal a zinc-binding s...

The S100 family consists of small acidic proteins, belonging to the EF-hand class of calcium-binding proteins. They are primarily regulatory proteins, involved in cell growth, cell structure regulation and signal transduction. Psoriasin (S100A7) is an 11.7 kDa protein that is highly upregulated in the epidermis of patients suffering from the chroni...

Methylotrophic bacteria can utilize C1 compounds as the sole source of carbon and energy. Oxidation of methylamine in these organisms is catalyzed by methylamine dehydrogenase (MADH), an inducible, periplasmic enzyme. This enzyme is a quinoprotein and contains the novel cofactor tryptophan tryptophylquinone (TTQ) (Fig. 1), which is derived from two...

Enzymatic and electron transfer activities have been studied by polarized absorption spectroscopy in single crystals of both binary and ternary complexes of methylamine dehydrogenase (MADH) with its redox partners. Within the crystals, MADH oxidizes methylamine, and the electrons are passed from the reduced tryptophan tryptophylquinone (TTQ) cofact...