Stephen Douthwaite

Stephen Douthwaite
University of Southern Denmark | SDU · Department of Biochemistry and Molecular Biology

PhD

About

150
Publications
28,471
Reads
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8,086
Citations
Additional affiliations
Position
  • Institut Pasteur
January 1996 - December 1997
March 1987 - present
University of Southern Denmark
Position
  • Professor Molecular Microbiology
Description
  • Ribosome-targeting antibiotics; resistance mechanisms conferred by nucleotide-specific rRNA methyltransferases; transfer of resistance determinants in Gram-negative pathogens.
Education
January 1977 - December 1981
Max-Planck-Institut für molekulare Genetik and Århus University
Field of study
  • Ribosomal RNA

Publications

Publications (150)
Article
Full-text available
Infection with Campylobacter jejuni is the major cause of human gastroenteritis in the United States and Europe, leading to debilitating autoimmune sequelae in many cases. While considerable progress has been made in detailing the infectious cycle of C. jejuni, a full understanding of the molecular mechanisms responsible for virulence remains to be...
Article
Full-text available
Dihydrouridine (D), a prevalent and evolutionarily conserved base in the transcriptome, primarily resides in tRNAs and, to a lesser extent, in mRNAs. Notably, this modification is found at position 2449 in the Escherichia coli 23S rRNA, strategically positioned near the ribosome’s peptidyl transferase site. Despite the prior identification, in E. c...
Preprint
Full-text available
Infection with Campylobacter jejuni is the major cause of human gastroenteritis in the United States and Europe, leading in many cases to debilitating autoimmune sequelae. While considerable progress has been made in detailing the infectious cycle of C. jejuni, a full understanding of the molecular mechanisms responsible for virulence remains to be...
Article
Full-text available
Campylobacter jejuni is a major cause of food poisoning worldwide, and remains the main infective agent in gastroenteritis and related intestinal disorders in Europe and the USA. As with all bacterial infections, the stages of adhesion to host tissue, survival in the host and eliciting disease all require the synthesis of proteinaceous virulence fa...
Article
Full-text available
The universal L-shaped tertiary structure of tRNAs is maintained with the help of nucleotide modifications within the D- and T-loops, and these modifications are most extensive within hyperthermophilic species. The obligate-commensal Nanoarchaeum equitans and its phylogenetically-distinct host Ignicoccus hospitalis grow physically coupled under ide...
Article
Full-text available
The C5-methylation of uracil to form 5-methyluracil (m 5 U) is a ubiquitous base modification of nucleic acids. Four enzyme families have converged to catalyze this methylation using different chemical solutions. Here, we investigate the evolution of 5-methyluracil synthase families in Mollicutes, a class of bacteria that has undergone extensive ge...
Preprint
Full-text available
The C5-methylation of uracil to form 5-methyluracil (m5U) is a ubiquitous base modification of nucleic acids. Four enzyme families have converged to catalyze this methylation using different chemical solutions. Here, we investigate the evolution of 5-methyluracil synthase families in Mollicutes, a class of bacteria that has undergone extensive geno...
Article
Full-text available
Campylobacter jejuni is a bacterial pathogen that is generally acquired as a zoonotic infection from poultry and animals. Adhesion of C. jejuni to human colorectal epithelial cells is weakened after loss of its cj0588 gene. The Cj0588 protein belongs to the type I group of TlyA (TlyAI) enzymes, which 2'‐O‐methylate nucleotide C1920 in 23S rRNA. Sli...
Article
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Objectives: To determine the mechanism of induction of erm(47) and its atypical expression in the Gram-positive opportunistic pathogen Helcococcus kunzii, where it confers resistance to a subset of clinically important macrolide, lincosamide and streptogramin B (MLSB) antibiotics. Methods: The resistant H. kunzii clinical isolate UCN99 was chall...
Article
Full-text available
Strains of the Pasteurellaceae bacteria Pasteurella multocida and Mannheimia haemolytica are major etiological agents of bovine respiratory disease (BRD). Treatment of BRD with antimicrobials is becoming more challenging due to the increasing occurrence of resistance in infecting strains. In Pasteurellaceae strains exhibiting resistance to multiple...
Article
Full-text available
The chemically mutagenized Escherichia coli strain AS19 was isolated on the basis of its enhanced sensitivity to different antibiotics, in particular to actinomycin. The strain was later modified to study rRNA modifications that confer antibiotic resistance. Here, we present the genome sequence of the variant E. coli AS19-RrmA ⁻ .
Article
Full-text available
The chemically mutagenized Escherichia coli strain AS19 was isolated on the basis of its enhanced sensitivity to different antibiotics, in particular to actinomy-cin. The strain was later modified to study rRNA modifications that confer antibiotic resistance. Here, we present the genome sequence of the variant E. coli AS19-RrmA-. E scherichia coli...
Article
Full-text available
Numerous bacterial pathogens express an ortholog of the enzyme TlyA, which is an rRNA 2′-O-methyltransferase associated with resistance to cyclic peptide antibiotics such as capreomycin. Several other virulence traits have also been attributed to TlyA, and these appear to be unrelated to its methyltransferase activity. The bacterial pathogen Campyl...
Article
Full-text available
In all free-living organisms a late-stage checkpoint in the biogenesis of the small ribosomal subunit involves rRNA modification by an RsmA/Dim1 methyltransferase. The hyperthermophilic archaeon Nanoarchaeum equitans, whose existence is confined to the surface of a second archaeon, Ignicoccus hospitalis, lacks an RsmA/Dim1 homolog. We demonstrate h...
Article
Full-text available
Mitochondrial dysfunction is a well-established cause of sensorineural deafness, but the pathophysiological events are poorly understood. Non-syndromic deafness and predisposition to aminoglycoside-induced deafness can be caused by specific mutations in the 12S rRNA gene of mtDNA and are thus maternally inherited traits. The pathophysiology induced...
Article
Full-text available
Significance Studies of antibiotic resistance are usually initiated in earnest only after resistance has become established in clinical pathogens. Here, we forewarn of a resistance mechanism to the novel antibiotics ketolides, which are only coming into broad medical practice. We show that the balanced activities and coordinated expression of two g...
Article
Full-text available
Objectives: To determine how resistance to macrolides is conferred in field isolates of Pasteurella multocida and Mannheimia haemolytica that lack previously identified resistance determinants for rRNA methylation, efflux and macrolide-modifying enzymes. Methods: Isolates of P. multocida and M. haemolytica identified as being highly resistant (M...
Article
Full-text available
Efficient protein synthesis in all organisms requires the post-transcriptional methylation of specific ribosomal ribonucleic acid (rRNA) and transfer RNA (tRNA) nucleotides. The methylation reactions are almost invariably catalyzed by enzymes that use S-adenosylmethionine (AdoMet) as the methyl group donor. One noteworthy exception is seen in some...
Article
Full-text available
Aminoglycoside antibiotics remain the drugs of choice for treatment of Pseudomonas aeruginosa infections, particularly for respiratory complications in cystic-fibrosis patients. Previous studies on other bacteria have shown that aminoglycosides have their primary target within the decoding region of 16S rRNA helix 44 with a secondary target in 23S...
Article
Full-text available
Objectives: 16S rRNA methyltransferases are an emerging mechanism conferring high-level resistance to clinically relevant aminoglycosides and have been associated with important mechanisms such as NDM-1. We sought genes encoding these enzymes in isolates highly resistant (MIC >200 mg/L) to gentamicin and amikacin from an Indian hospital and we add...
Article
Full-text available
Tildipirosin is a 16-membered-ring macrolide developed to treat bacterial pathogens, including Mannheimia haemolytica and Pasteurella multocida, that cause respiratory tract infections in cattle and swine. Here we evaluated the efficacy of tildipirosin at inhibiting protein synthesis on the ribosome (50% inhibitory concentration [IC50], 0.23 ± 0.01...
Article
Full-text available
The binding site of the cyclic peptide antibiotics capreomycin and viomycin is located on the ribosomal subunit interface close to nucleotides C1409 in 16S rRNA and C1920 in 23S rRNA. In Mycobacterium tuberculosis, the 2'-hydroxyls of both nucleotides are methylated by the enzyme TlyA. Loss of these methylations through inactivation of TlyA confers...
Article
Full-text available
The bacterial pathogens Mannheimia haemolytica and Pasteurella multocida are major etiological agents in respiratory tract infections of cattle. Although these infections can generally be successfully treated with veterinary macrolide antibiotics, a few recent isolates have shown resistance to these drugs. Macrolide resistance in members of the fam...
Article
Full-text available
The veterinary antibiotic tildipirosin (20,23-dipiperidinyl-mycaminosyl-tylonolide, Zuprevo) was developed recently to treat bovine and swine respiratory tract infections caused by bacterial pathogens such as Pasteurella multocida. Tildipirosin is a derivative of the naturally occurring compound tylosin. Here, we define drug-target interactions by...
Article
Full-text available
The 23S rRNA nucleotide m2G2445 is highly conserved in bacteria, and in Escherichia coli this modification is added by the enzyme YcbY. With lengths of around 700 amino acids, YcbY orthologs are the largest rRNA methyltransferases identified in Gram-negative bacteria, and they appear to be fusions from two separate proteins found in Gram-positives....
Article
Full-text available
Respiratory tract infections in cattle are commonly associated with the bacterial pathogens Mannheimia haemolytica and Pasteurella multocida. These infections can generally be successfully treated in the field with one of several groups of antibiotics, including macrolides. A few recent isolates of these species exhibit resistance to veterinary mac...
Article
Full-text available
Methyltransferases that use S-adenosylmethionine (AdoMet) as a cofactor to catalyse 5-methyl uridine (m5U) formation in tRNAs and rRNAs are widespread in Bacteria and Eukaryota, and are also found in certain Archaea. These enzymes belong to the COG2265 cluster, and the Gram-negative bacterium Escherichia coli possesses three paralogues. These compr...
Conference Paper
Full-text available
The stable RNAs (tRNAs and rRNAs) of all organisms are post-transcriptionally modified to improve their functions in protein synthesis (Grosjean, 2005). The tRNAs exhibit the densest concentration of modifications with generally about 10% of their nucleotides being modified. In Escherichia colitRNAs, thirty-one distinct types of modified nucleotide...
Article
Full-text available
Erm methyltransferases are prevalent in pathogenic bacteria and confer resistance to macrolide, lincosamide, and streptogramin B antibiotics by specifically methylating the 23S ribosomal RNA at nucleotide A2058. We have identified motifs within the rRNA substrate that are required for methylation by Erm. Substrate molecules were constructed in a co...
Article
Full-text available
Mannheimia haemolytica and Pasteurella multocida are aetiological agents commonly associated with respiratory tract infections in cattle. Recent isolates of these pathogens have been shown to be resistant to macrolides and other ribosome-targeting antibiotics. Direct analysis of the 23S rRNAs by mass spectrometry revealed that nucleotide A2058 is m...
Article
Full-text available
Aminoglycosides are ribosome-targeting antibiotics and a major drug group of choice in the treatment of serious enterococcal infections. Here we show that aminoglycoside resistance in Enterococcus faecium strain CIP 54-32 is conferred by the chromosomal gene efmM, encoding the E. faecium methyltransferase, as well as by the previously characterized...
Article
Full-text available
Ribosomal RNAs fold into phylogenetically conserved secondary and tertiary structures that determine their function in protein synthesis. We have investigated Escherichia coli 23S rRNA to identify structural elements that interact with antibiotic and protein ligands. Using a combination of molecular genetic and biochemical probing techniques, we ha...
Article
Full-text available
Transfer RNAs are the most densely modified nucleic acid molecules in living cells. In Escherichia coli, more than 30 nucleoside modifications have been characterized, ranging from methylations and pseudouridylations to more complex additions that require multiple enzymatic steps. Most of the modifying enzymes have been identified, although a few n...
Article
Full-text available
Methyltransferase enzymes that use S-adenosylmethionine as a cofactor to catalyze 5-methyl uridine (m(5)U) formation in tRNAs and rRNAs are widespread in Bacteria and Eukaryota, but are restricted to the Thermococcales and Nanoarchaeota groups amongst the Archaea. The RNA m(5)U methyltransferases appear to have arisen in Bacteria and were then disp...
Article
Full-text available
Ribosome-targeting antibiotics block protein synthesis by binding at functionally important regions of the bacterial rRNA. Resistance is often conferred by addition of a methyl group at the antibiotic binding site within an rRNA region that is already highly modified with several nucleotide methylations. In bacterial rRNA, each methylation requires...
Article
Full-text available
The rRNAs of Escherichia coli contain four 2'-O-methylated nucleotides. Similar to other bacterial species and in contrast with Archaea and Eukaryota, the E. coli rRNA modifications are catalysed by specific methyltransferases that find their nucleotide targets without being guided by small complementary RNAs. We show here that the ygdE gene encode...
Article
Many types of chemically diverse antibiotics inhibit bacterial growth by binding to the ribosome and blocking protein synthesis. The large majority of these antibiotics have a distinct target site on either the 30S or the 50S ribosomal subunit. Thus, these antibiotics are generally specific inhibitors of steps in protein synthesis that are associat...
Article
Full-text available
Pseudouridines in the stable RNAs of Bacteria are seldom subjected to further modification. There are 11 pseudouridine (Psi) sites in Escherichia coli rRNA, and further modification is found only at Psi1915 in 23S rRNA, where the N-3 position of the base becomes methylated. Here, we report the identity of the E. coli methyltransferase that specific...
Article
Full-text available
Methylation at the 5-position of cytosine [m(5)C (5-methylcytidine)] occurs at three RNA nucleotides in Escherichia coli. All these modifications are at highly conserved nucleotides in the rRNAs, and each is catalyzed by its own m(5)C methyltransferase enzyme. Two of the enzymes, RsmB and RsmF, are already known and methylate 16S rRNA at nucleotide...
Article
Full-text available
Methylation is the most common RNA modification in the three domains of life. Transfer of the methyl group from S-adenosyl-l-methionine (AdoMet) to specific atoms of RNA nucleotides is catalyzed by methyltransferase (MTase) enzymes. The rRNA MTase RlmI (rRNA large subunit methyltransferase gene I; previously known as YccW) specifically modifies Esc...
Article
Full-text available
RlmA(II) methylates the N1-position of nucleotide G748 in hairpin 35 of 23 S rRNA. The resultant methyl group extends into the peptide channel of the 50 S ribosomal subunit and confers resistance to tylosin and other mycinosylated macrolide antibiotics. Methylation at G748 occurs in several groups of Gram-positive bacteria, including the tylosin-pr...
Article
Full-text available
Resistance to antibiotics that target the bacterial ribosome is often conferred by methylation at specific nucleotides in the rRNA. The nucleotides that become methylated are invariably key sites of antibiotic interaction. The addition of methyl groups to each of these nucleotides is catalyzed by a specific methyltransferase enzyme. The Erm methylt...
Article
Full-text available
Translocation during the elongation phase of protein synthesis involves the relative movement of the 30S and 50S ribosomal subunits. This movement is the target of tuberactinomycin antibiotics. Here, we describe the isolation and characterization of mutants of Thermus thermophilus selected for resistance to the tuberactinomycin antibiotic capreomyc...
Article
Full-text available
A telithromycin-resistant clinical isolate of Streptococcus pneumoniae (strain P1501016) has been found to contain a version of erm(B) that is altered by a 136-bp deletion in the leader sequence. By allele replacement mutagenesis, a second strain of S. pneumoniae (PC13) with a wild-type erm(B) gene was transformed to the telithromycin-resistant phe...
Article
Full-text available
The methyltransferase RlmA(II) (formerly TlrB) is found in many Gram-positive bacteria, and methylates the N-1 position of nucleotide G748 within the loop of hairpin 35 in 23S rRNA. Methylation of the rRNA by RlmA(II) confers resistance to tylosin and other mycinosylated 16-membered ring macrolide antibiotics. We have previously solved the solution...
Article
Full-text available
Members of the archease superfamily of proteins are represented in all three domains of life. Archease genes are generally located adjacent to genes encoding proteins involved in DNA or RNA processing. Archease have therefore been predicted to play a modulator or chaperone role in selected steps of DNA or RNA metabolism, although the roles of arche...
Article
Full-text available
Posttranscriptional modifications on the base or sugar of ribonucleosides generally result in mass increases that can be measured by mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a direct and accurate means of determining the masses of RNAs. Mass spectra produced by MALDI are relatively straightforwa...
Article
Full-text available
The methyltransferase RsmG methylates the N7 position of nucleotide G535 in 16S rRNA of Bacillus subtilis (corresponding to G527 in Escherichia coli). Disruption of rsmG resulted in low-level resistance to streptomycin. A growth competition assay revealed that there are no differences in fitness between the rsmG mutant and parent strains under the...
Article
The cyclic peptide antibiotics capreomycin and viomycin are generally effective against the bacterial pathogen Mycobacterium tuberculosis. However, recent virulent isolates have become resistant by inactivation of their tlyA gene. We show here that tlyA encodes a 2'-O-methyltransferase that modifies nucleotide C1409 in helix 44 of 16S rRNA and nucl...
Article
Full-text available
Nucleotide methylations are the most common type of rRNA modification in bacteria, and are introduced post-transcriptionally by a wide variety of site-specific enzymes. Three 5-methylcytidine (m(5)C) bases are found in the rRNAs of Escherichia coli and one of these, at nucleotide 1407 in 16 S rRNA, is the modification product of the methyltransfera...