Imperial College London

Professor Angelika Gründling

Faculty of MedicineDepartment of Infectious Disease

Professor of Molecular Microbiology
 
 
 
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Contact

 

+44 (0)20 7594 5256a.grundling Website

 
 
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Location

 

3.21Flowers buildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
to

75 results found

Grundling A, Collet J-F, 2021, Editorial overview: "All in all, it is not just another brick in the wall": new concepts and mechanisms on how bacteria build their wall, CURRENT OPINION IN MICROBIOLOGY, Vol: 62, Pages: 110-113, ISSN: 1369-5274

Journal article

Rismondo J, Gillis A, Grundling A, 2021, Modifications of cell wall polymers in Gram-positive bacteria by multi-component transmembrane glycosylation systems, Current Opinion in Microbiology, Vol: 60, Pages: 24-33, ISSN: 1369-5274

Secondary cell wall polymers fulfil diverse and important functions within the cell wall of Gram-positive bacteria. Here, we will provide a brief overview of the principles of teichoic acid and complex secondary cell wall polysaccharide biosynthesis pathways in Firmicutes and summarize the recently revised mechanism for the decoration of teichoic acids with d-alanines. Many cell wall polymers are decorated with glycosyl groups, either intracellularly or extracellularly. The main focus of this review will be on the extracellular glycosylation mechanism and recent advances that have been made in the identification of enzymes involved in this process. Based on the proteins involved, we propose to rename the system to multi-component transmembrane glycosylation system in place of three-component glycosylation system.

Journal article

Wu C-H, Rismondo J, Morgan RML, Shen Y, Loessner MJ, Larrouy-Maumus G, Freemont PS, Grundling Aet al., 2021, Bacillus subtilis YngB contributes to wall teichoic acid glucosylation and glycolipid formation during anaerobic growth, Journal of Biological Chemistry, Vol: 296, Pages: 1-14, ISSN: 0021-9258

UTP-glucose-1-phosphate uridylyltransferases are enzymes that produce UDP-glucose from UTP and glucose-1-phosphate. In Bacillus subtilis 168, UDP-glucose is required for the decoration of wall teichoic acid (WTA) with glucose residues and the formation of glucolipids. The B. subtilis UGPase GtaB is essential for UDP-glucose production under standard aerobic growth conditions, and gtaB mutants display severe growth and morphological defects. However, bioinformatics predictions indicate that two other UTP-glucose-1-phosphate uridylyltransferases are present in B. subtilis. Here, we investigated the function of one of them named YngB. The crystal structure of YngB revealed that the protein has the typical fold and all necessary active site features of a functional UGPase. Furthermore, UGPase activity could be demonstrated in vitro using UTP and glucose-1-phosphate as substrates. Expression of YngB from a synthetic promoter in a B. subtilis gtaB mutant resulted in the reintroduction of glucose residues on WTA and production of glycolipids, demonstrating that the enzyme can function as UGPase in vivo. When WT and mutant B. subtilis strains were grown under anaerobic conditions, YngB-dependent glycolipid production and glucose decorations on WTA could be detected, revealing that YngB is expressed from its native promoter under anaerobic condition. Based on these findings, along with the structure of the operon containing yngB and the transcription factor thought to be required for its expression, we propose that besides WTA, potentially other cell wall components might be decorated with glucose residues during oxygen-limited growth condition.

Journal article

Rismondo J, Schulz LM, Yacoub M, Wadhawan A, Hoppert M, Dionne MS, Gründling Aet al., 2021, EslB is required for cell wall biosynthesis and modification in Listeria monocytogenes., Journal of Bacteriology, Vol: 203, Pages: 1-16, ISSN: 0021-9193

Lysozyme is an important component of the innate immune system. It functions by hydrolysing the peptidoglycan (PG) layer of bacteria. The human pathogen Listeria monocytogenes is intrinsically lysozyme resistant. The peptidoglycan N-deacetylase PgdA and O-acetyltransferase OatA are two known factors contributing to its lysozyme resistance. Furthermore, it was shown that the absence of components of an ABC transporter, here referred to as EslABC, leads to reduced lysozyme resistance. How its activity is linked to lysozyme resistance is still unknown. To investigate this further, a strain with a deletion in eslB, coding for a membrane component of the ABC transporter, was constructed in L. monocytogenes strain 10403S. The eslB mutant showed a 40-fold reduction in the minimal inhibitory concentration to lysozyme. Analysis of the PG structure revealed that the eslB mutant produced PG with reduced levels of O-acetylation. Using growth and autolysis assays, we show that the absence of EslB manifests in a growth defect in media containing high concentrations of sugars and increased endogenous cell lysis. A thinner PG layer produced by the eslB mutant under these growth conditions might explain these phenotypes. Furthermore, the eslB mutant had a noticeable cell division defect and formed elongated cells. Microscopy analysis revealed that an early cell division protein still localized in the eslB mutant indicating that a downstream process is perturbed. Based on our results, we hypothesize that EslB affects the biosynthesis and modification of the cell wall in L. monocytogenes and is thus important for the maintenance of cell wall integrity.IMPORTANCE The ABC transporter EslABC is associated with the intrinsic lysozyme resistance of Listeria monocytogenes However, the exact role of the transporter in this process and in the physiology of L. monocytogenes is unknown. Using different assays to characterize an eslB deletion strain, we found that the absence of EslB not only af

Journal article

Zhang K, Raju C, Zhong W, Pethe K, Gründling A, Chan-Park MBet al., 2021, Cationic glycosylated block Co-β-peptide acts on the cell wall of gram-positive bacteria as anti-biofilm agents, ACS Applied Bio Materials, Vol: 4, Pages: 3749-3761, ISSN: 2576-6422

Antimicrobial resistance is a global threat. In addition to the emergence of resistance to last resort drugs, bacteria escape antibiotics killing by forming complex biofilms. Strategies to tackle antibiotic resistance as well as biofilms are urgently needed. Wall teichoic acid (WTA), a generic anionic glycopolymer present on the cell surface of many Gram-positive bacteria, has been proposed as a possible therapeutic target, but its druggability remains to be demonstrated. Here we report a cationic glycosylated block co-β-peptide that binds to WTA. By doing so, the co-β-peptide not only inhibits biofilm formation, it also disperses preformed biofilms in several Gram-positive bacteria and resensitizes methicillin-resistant Staphylococcus aureus to oxacillin. The cationic block of the co-β-peptide physically interacts with the anionic WTA within the cell envelope, whereas the glycosylated block forms a nonfouling corona around the bacteria. This reduces physical interaction between bacteria-substrate and bacteria-biofilm matrix, leading to biofilm inhibition and dispersal. The WTA-targeting co-β-peptide is a promising lead for the future development of broad-spectrum anti-biofilm strategies against Gram-positive bacteria.

Journal article

Zhong W, Shi Z, Mahadevegowda SH, Liu B, Zhang K, Hui Koh C, Ruan L, Chen Y, Zeden MS, Pee CJE, Marimuthu K, Pratim De P, Tek Ng O, Zhu Y, Chi YR, Hammond PT, Yang L, Gan Y-H, Pethe K, Greenberg EP, Grundling A, Chan-Park MBet al., 2020, Designer broad-spectrum polyimidazolium antibiotics, Proceedings of the National Academy of Sciences of USA, Vol: 117, Pages: 31376-31385, ISSN: 0027-8424

For a myriad of different reasons most antimicrobial peptides (AMPs) have failed to reach clinical application. Different AMPs have different shortcomings including but not limited to toxicity issues, potency, limited spectrum of activity, or reduced activity in situ. We synthesized several cationic peptide mimics, main-chain cationic polyimidazoliums (PIMs), and discovered that, although select PIMs show little acute mammalian cell toxicity, they are potent broad-spectrum antibiotics with activity against even pan-antibiotic-resistant gram-positive and gram-negative bacteria, and mycobacteria. We selected PIM1, a particularly potent PIM, for mechanistic studies. Our experiments indicate PIM1 binds bacterial cell membranes by hydrophobic and electrostatic interactions, enters cells, and ultimately kills bacteria. Unlike cationic AMPs, such as colistin (CST), PIM1 does not permeabilize cell membranes. We show that a membrane electric potential is required for PIM1 activity. In laboratory evolution experiments with the gram-positive Staphylococcus aureus we obtained PIM1-resistant isolates most of which had menaquinone mutations, and we found that a site-directed menaquinone mutation also conferred PIM1 resistance. In similar experiments with the gram-negative pathogen Pseudomonas aeruginosa, PIM1-resistant mutants did not emerge. Although PIM1 was efficacious as a topical agent, intraperitoneal administration of PIM1 in mice showed some toxicity. We synthesized a PIM1 derivative, PIM1D, which is less hydrophobic than PIM1. PIM1D did not show evidence of toxicity but retained antibacterial activity and showed efficacy in murine sepsis infections. Our evidence indicates the PIMs have potential as candidates for development of new drugs for treatment of pan-resistant bacterial infections.

Journal article

Wu C-H, Rismondo J, Morgan RML, Shen Y, Loessner MJ, Larrouy-Maumus G, Freemont PS, Gründling Aet al., 2020, Bacillus subtilis YngB contributes to wall teichoic acid glucosylation and glycolipid formation during anaerobic growth, Publisher: Cold Spring Harbor Laboratory

<jats:title>Abstract</jats:title><jats:p>UTP-glucose-1-phosphate uridylyltransferases (UGPases) are enzymes that produce UDP-glucose from UTP and glucose-1-phosphate. In <jats:italic>Bacillus subtilis</jats:italic> 168, UDP-glucose is required for the decoration of wall teichoic acid (WTA) with glucose residues and the formation of glucolipids. The <jats:italic>B. subtilis</jats:italic> UGPase GtaB is essential for UDP-glucose production under standard aerobic growth conditions, and <jats:italic>gtaB</jats:italic> mutants display severe growth and morphological defects. However, bioinformatics predictions indicate that two other UGPases, are present in <jats:italic>B. subtilis</jats:italic>. Here, we investigated the function of one of them named YngB. The crystal structure of YngB revealed that the protein has the typical fold and all necessary active site features of a functional UGPase. Furthermore, UGPase activity could be demonstrated <jats:italic>in vitro</jats:italic> using UTP and glucose-1-phosphate as substrates. Expression of YngB from a synthetic promoter in a <jats:italic>B. subtilis gtaB</jats:italic> mutant resulted in the reintroduction of glucose residues on WTA and production of glycolipids, demonstrating that the enzyme can function as UGPase <jats:italic>in vivo</jats:italic>. When wild-type and mutant <jats:italic>B. subtilis</jats:italic> strains were grown under anaerobic conditions, YngB-dependent glycolipid production and glucose decorations on WTA could be detected, revealing that YngB is expressed from its native promoter under anaerobic condition. Based on these findings, along with the structure of the operon containing <jats:italic>yngB</jats:italic> and the transcription factor thought to be required for its expression, we propose that besides WTA, potentially other cell wall components might be decorated with g

Working paper

Rismondo J, Haddad TFM, Shen Y, Loessner MJ, Grundling Aet al., 2020, GtcA is required for LTA glycosylation in Listeria monocytogenes serovar 1/2a and Bacillus subtilis, The Cell Surface, Vol: 6, ISSN: 2468-2330

The cell wall polymers wall teichoic acid (WTA) and lipoteichoic acid (LTA) are often modified with glycosyl and D-alanine residues. Recent studies have shown that a three-component glycosylation system is used for the modification of LTA in several Gram-positive bacteria including Bacillus subtilis and Listeria monocytogenes. In the L. monocytogenes 1/2a strain 10403S, the cytoplasmic glycosyltransferase GtlA is thought to use UDP-galactose to produce the C55-P-galactose lipid intermediate, which is transported across the membrane by an unknown flippase. Next, the galactose residue is transferred onto the LTA backbone on the outside of the cell by the glycosyltransferase GtlB. Here we show that GtcA is necessary for the glycosylation of LTA in L. monocytogenes 10403S and B. subtilis 168 and we hypothesize that these proteins act as C55-P-sugar flippases. With this we revealed that GtcA is involved in the glycosylation of both teichoic acid polymers in L. monocytogenes 10403S, namely WTA with N-acetylglucosamine and LTA with galactose residues. These findings indicate that the L. monocytogenes GtcA protein can act on different C55-P-sugar intermediates. Further characterization of GtcA in L. monocytogenes led to the identification of residues essential for its overall function as well as residues, which predominately impact WTA or LTA glycosylation.

Journal article

Walter A, Unsleber S, Rismondo J, Jorge AM, Peschel A, Grundling A, Mayer Cet al., 2020, Phosphoglycerol-type wall and lipoteichoic acids are enantiomeric polymers differentiated by the stereospecific glycerophosphodiesterase GlpQ (vol 295, pg 4024, 2020), JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 295, Pages: 8873-8873, ISSN: 0021-9258

Journal article

Zeden MS, Kviatkovski I, Schuster CF, Thomas VC, Fey PD, Grundling Aet al., 2020, Identification of the main glutamine and glutamate transporters in Staphylococcus aureus and their impact on c-di-AMP production, Molecular Microbiology, Vol: 113, Pages: 1085-1100, ISSN: 0950-382X

A Staphylococcus aureus strain deleted for the c‐di‐AMP cyclase gene dacA is unable to survive in rich medium unless it acquires compensatory mutations. Previously identified mutations were in opuD, encoding the main glycine‐betaine transporter, and alsT, encoding a predicted amino acid transporter. Here, we show that inactivation of OpuD restores the cell size of a dacA mutant to near wild‐type (WT) size, while inactivation of AlsT does not. AlsT was identified as an efficient glutamine transporter, indicating that preventing glutamine uptake in rich medium rescues the growth of the S. aureus dacA mutant. In addition, GltS was identified as a glutamate transporter. By performing growth curves with WT, alsT and gltS mutant strains in defined medium supplemented with ammonium, glutamine or glutamate, we revealed that ammonium and glutamine, but not glutamate promote the growth of S. aureus. This suggests that besides ammonium also glutamine can serve as a nitrogen source under these conditions. Ammonium and uptake of glutamine via AlsT and hence likely a higher intracellular glutamine concentration inhibited c‐di‐AMP production, while glutamate uptake had no effect. These findings provide, besides the previously reported link between potassium and osmolyte uptake, a connection between nitrogen metabolism and c‐di‐AMP signalling in S. aureus.

Journal article

Schuster CF, Wiedemann DM, Kirsebom FCM, Santiago M, Walker S, Gründling Aet al., 2020, High‐throughput transposon sequencing highlights the cell wall as an important barrier for osmotic stress in methicillin resistant Staphylococcus aureus and underlines a tailored response to different osmotic stressors, Molecular Microbiology, Vol: 113, Pages: 699-717, ISSN: 0950-382X

Staphylococcus aureus is an opportunistic pathogen that can cause soft tissue infections but is also a frequent cause of foodborne illnesses. One contributing factor for this food association is its high salt tolerance allowing this organism to survive commonly used food preservation methods. How this resistance is mediated is poorly understood, particularly during long‐term exposure. In this study, we used TN‐seq to understand how the responses to osmotic stressors differ. Our results revealed distinctly different long‐term responses to NaCl, KCl and sucrose stresses. In addition, we identified the DUF2538 domain containing gene SAUSA300_0957 (gene 957) as essential under salt stress. Interestingly, a 957 mutant was less susceptible to oxacillin and showed increased peptidoglycan crosslinking. The salt sensitivity phenotype could be suppressed by amino acid substitutions in the transglycosylase domain of the penicillin binding protein Pbp2, and these changes restored the peptidoglycan crosslinking to WT levels. These results indicate that increased crosslinking of the peptidoglycan polymer can be detrimental and highlight a critical role of the bacterial cell wall for osmotic stress resistance. This study will serve as a starting point for future research on osmotic stress response and help develop better strategies to tackle foodborne staphylococcal infections.

Journal article

Walter A, Unsleber S, Rismondo J, Jorge AM, Peschel A, Grundling A, Mayer Cet al., 2020, Phosphoglycerol-type wall- and lipoteichoic acids are enantiomeric polymersdifferentiated by the stereospecific glycerophosphodiesterase GlpQ, Journal of Biological Chemistry, Vol: 12, Pages: 4024-4034, ISSN: 0021-9258

The cell envelope of Gram-positive bacteria generally comprises two types of polyanionic polymers linked to either peptidoglycan (wall teichoic acids; WTA) or to membrane glycolipids (lipoteichoic acids; LTA). In some bacteria, including Bacillus subtilis strain 168, both WTA and LTA are glycerolphosphate polymers yet are synthesized through different pathways and have distinct but incompletely understood morphogenetic functions during cell elongation and division. We show here that the exolytic sn-glycerol-3-phosphodiesterase GlpQ can discriminate between B. subtilis WTA and LTA. GlpQ completely degraded unsubstituted WTA, which lacks substituents at the glycerol residues, by sequentially removing glycerolphosphates from the free end of the polymer up to the peptidoglycan linker. In contrast, GlpQ could not degrade unsubstituted LTA unless it was partially precleaved, allowing access of GlpQ to the other end of the polymer, which, in the intact molecule, is protected by a connection to the lipid anchor. Differences in stereochemistry between WTA and LTA have been suggested previously on the basis of differences in their biosynthetic precursors and chemical degradation products. The differential cleavage of WTA and LTA by GlpQ reported here represents the first direct evidence that they are enantiomeric polymers: WTA is made of sn-glycerol-3-phosphate, and LTA is made of sn-glycerol-1-phosphate. Their distinct stereochemistries reflect the dissimilar physiological and immunogenic properties of WTA and LTA. It also enables differential degradation of the two polymers within the same envelope compartment in vivo, particularly under phosphate-limiting conditions, when B. subtilis specifically degrades WTA and replaces it with phosphate-free teichuronic acids.

Journal article

Sumrall ET, Schefer CRE, Rismondo J, Grundling A, Boulos S, Schneider SR, Loessner MJ, Shen Yet al., 2020, Galactosylated wall-teichoic acid, but not lipoteichoic acid, retains InlB on the surface of serovar 4b Listeria monocytogenes, Molecular Microbiology, Vol: 113, Pages: 638-649, ISSN: 0950-382X

Listeria monocytogenes is a Gram-positive, intracellular pathogen harboring the surface-associated virulence factor InlB, which enables entry into certain host cells. Structurally diverse wall-teichoic acids (WTAs), which can also be differentially glycosylated, determine the antigenic basis of the various Listeria serovars. WTAs have many physiological functions; they can serve as receptors for bacteriophages, and provide a substrate for binding of surface proteins such as InlB. In contrast, the membrane-anchored lipoteichoic acids (LTAs) do not show significant variation and do not contribute to serovar determination. It was previously demonstrated that surface-associated InlB non-covalently adheres to both WTA and LTA, mediating its retention on the cell wall. Here, we demonstrate that in a highly virulent serovar 4b strain, two genes gtlB and gttB are responsible for galactosylation of LTA and WTA, respectively. We evaluated the InlB surface retention in mutants lacking each of these two genes, and found that only galactosylated WTA is required for InlB surface presentation and function, cellular invasiveness, and phage adsorption, while galactosylated LTA plays no role thereof. Our findings demonstrate that a simple pathogen-defining serovar antigen, that mediates bacteriophage susceptibility, is necessary and sufficient to sustain the function of an important virulence factor.

Journal article

Rismondo J, Schulz LM, Yacoub M, Wadhawan A, Hoppert M, Dionne MS, Gründling Aet al., 2020, EslB is required for cell wall biosynthesis and modification inListeria monocytogenes, Publisher: Cold Spring Harbor Laboratory

<jats:title>ABSTRACT</jats:title><jats:p>Lysozyme is an important component of the innate immune system. It functions by hydrolysing the peptidoglycan (PG) layer of bacteria. The human pathogen<jats:italic>Listeria monocytogenes</jats:italic>is intrinsically lysozyme resistant. The peptidoglycan<jats:italic>N</jats:italic>-deacetylase PgdA and<jats:italic>O</jats:italic>-acetyltransferase OatA are two known factors contributing to its lysozyme resistance. Furthermore, it was shown that the absence of components of an ABC transporter, here referred to as EslABC, leads to reduced lysozyme resistance. How its activity is linked to lysozyme resistance is still unknown. To investigate this further, a strain with a deletion in<jats:italic>eslB</jats:italic>, coding for a membrane component of the ABC transporter, was constructed in<jats:italic>L. monocytogenes</jats:italic>strain 10403S. The<jats:italic>eslB</jats:italic>mutant showed a 40-fold reduction in the minimal inhibitory concentration to lysozyme. Analysis of the PG structure revealed that the<jats:italic>eslB</jats:italic>mutant produced PG with reduced levels of<jats:italic>O</jats:italic>-acetylation. Using growth and autolysis assays, we show that the absence of EslB manifests in a growth defect in media containing high concentrations of sugars and increased endogenous cell lysis. A thinner PG layer produced by the<jats:italic>eslB</jats:italic>mutant under these growth conditions might explain these phenotypes. Furthermore, the<jats:italic>eslB</jats:italic>mutant had a noticeable cell division defect and formed elongated cells. Microscopy analysis revealed that an early cell division protein still localized in the<jats:italic>eslB</jats:italic>mutant indicating that a downstream process is perturbed. Based on our results, we hypothesize that EslB affects the b

Working paper

Walter A, Unsleber S, Rismondo J, Jorge AM, Peschel A, Gründling A, Mayer Cet al., 2020, Phosphoglycerol-type wall- and lipoteichoic acids are enantiomeric polymers differentially cleaved by the stereospecific glycerophosphodiesterase GlpQ

<jats:title>ABSTRACT</jats:title><jats:p>The cell envelope of Gram-positive bacteria generally comprises two types of polyanionic polymers, either linked to peptidoglycan, wall teichoic acids (WTA), or to membrane glycolipids, lipoteichoic acids (LTA). In some bacteria, including<jats:italic>Bacillus subtilis</jats:italic>strain 168, WTA and LTA both are glycerolphosphate polymers, yet are synthesized by different pathways and have distinct, although not entirely understood morphogenetic functions during cell elongation and division. We show here that the exo-lytic<jats:italic>sn</jats:italic>-glycerol-3-phosphodiesterase GlpQ can discriminate between<jats:italic>B. subtilis</jats:italic>WTA and LTA polymers. GlpQ completely degrades WTA, lacking modifications at the glycerol residues, by sequentially removing glycerolphosphates from the free end of the polymer up to the peptidoglycan linker. In contrast, GlpQ is unable to cleave unmodified LTA. LTA can only be hydrolyzed by GlpQ when the polymer is partially pre-cleaved, thereby allowing GlpQ to get access to the end of the polymer that is usually protected by a connection to the lipid anchor. This indicates that WTA and LTA are enantiomeric polymers: WTA is made of<jats:italic>sn</jats:italic>-glycerol-3-phosphate and LTA is made of<jats:italic>sn</jats:italic>-glycerol-1-phosphate. Differences in stereochemistry between WTA and LTA were assumed based on differences in biosynthesis precursors and chemical degradation products, but so far had not been demonstrated directly by differential, enantioselective cleavage of isolated polymers. The discriminative stereochemistry impacts the dissimilar physiological and immunogenic properties of WTA and LTA and enables independent degradation of the polymers, while appearing in the same location; e.g. under phosphate limitation,<jats:italic>B. subtilis</jats:italic>168 specifically hyd

Journal article

Rismondo J, Haddad TFM, Shen Y, Loessner MJ, Gründling Aet al., 2019, GtcA is required for LTA glycosylation inListeria monocytogenesserovar 1/2a andBacillus subtilis, Publisher: Cold Spring Harbor Laboratory

<jats:title>ABSTRACT</jats:title><jats:p>The cell wall polymers wall teichoic acid (WTA) and lipoteichoic acid (LTA) are often modified with glycosyl and D-alanine residues. Recent studies have shown that a three-component glycosylation system is used for the modification of LTA in several Gram-positive bacteria including<jats:italic>Bacillus subtilis</jats:italic>and<jats:italic>Listeria monocytogenes</jats:italic>. In the<jats:italic>L. monocytogenes</jats:italic>1/2a strain 10403S, the cytoplasmic glycosyltransferase GtlA is thought to use UDP-galactose to produce the C<jats:sub>55</jats:sub>-P-galactose lipid intermediate, which is transported across the membrane by an unknown flippase. Next, the galactose residue is transferred onto the LTA backbone on the outside of the cell by the glycosyltransferase GtlB. Here we show that GtcA is necessary for the glycosylation of LTA in<jats:italic>L. monocytogenes</jats:italic>10403S and<jats:italic>B. subtilis</jats:italic>168 and we hypothesize that these proteins act as C<jats:sub>55</jats:sub>-P-sugar flippases. With this we revealed that GtcA is involved in the glycosylation of both teichoic acid polymers in<jats:italic>L. monocytogenes</jats:italic>10403S, namely WTA with N-acetylglucosamine and LTA with galactose residues. These findings indicate that the<jats:italic>L. monocytogenes</jats:italic>GtcA protein can act on different C<jats:sub>55</jats:sub>-P-sugar intermediates. Further characterization of GtcA in<jats:italic>L. monocytogenes</jats:italic>led to the identification of residues essential for its overall function as well as residues, which predominately impact WTA or LTA glycosylation.</jats:p><jats:sec><jats:title>GRAPHICAL ABSTRACT</jats:title><jats:fig id="ufig1" position="anchor" orientation="portrait

Working paper

Sumrall ET, Shen Y, Keller AP, Rismondo J, Pavlou M, Eugster MR, Boulos S, Disson O, Thouvenot P, Kilcher S, Wollscheid B, Cabanes D, Lecuit M, Gründling A, Loessner MJet al., 2019, Phage resistance at the cost of virulence: Listeria monocytogenes serovar 4b requires galactosylated teichoic acids for InlB-mediated invasion, PLoS Pathogens, Vol: 15, ISSN: 1553-7366

The intracellular pathogen Listeria monocytogenes is distinguished by its ability to invade and replicate within mammalian cells. Remarkably, of the 15 serovars within the genus, strains belonging to serovar 4b cause the majority of listeriosis clinical cases and outbreaks. The Listeria O-antigens are defined by subtle structural differences amongst the peptidoglycan-associated wall-teichoic acids (WTAs), and their specific glycosylation patterns. Here, we outline the genetic determinants required for WTA decoration in serovar 4b L. monocytogenes, and demonstrate the exact nature of the 4b-specific antigen. We show that challenge by bacteriophages selects for surviving clones that feature mutations in genes involved in teichoic acid glycosylation, leading to a loss of galactose from both wall teichoic acid and lipoteichoic acid molecules, and a switch from serovar 4b to 4d. Surprisingly, loss of this galactose decoration not only prevents phage adsorption, but leads to a complete loss of surface-associated Internalin B (InlB),the inability to form actin tails, and a virulence attenuation in vivo. We show that InlB specifically recognizes and attaches to galactosylated teichoic acid polymers, and is secreted upon loss of this modification, leading to a drastically reduced cellular invasiveness. Consequently, these phage-insensitive bacteria are unable to interact with cMet and gC1q-R host cell receptors, which normally trigger cellular uptake upon interaction with InlB. Collectively, we provide detailed mechanistic insight into the dual role of a surface antigen crucial for both phage adsorption and cellular invasiveness, demonstrating a trade-off between phage resistance and virulence in this opportunistic pathogen.

Journal article

Zeden MS, Kviatkovski I, Schuster CF, Thomas VC, Fey PD, Gründling Aet al., 2019, Identification of the main glutamine and glutamate transporters inStaphylococcus aureusand their impact on c-di-AMP production

<jats:title>Summary</jats:title><jats:p>A<jats:italic>Staphylococcus aureus</jats:italic>strain deleted for the c-di-AMP cyclase gene<jats:italic>dacA</jats:italic>is unable to survive in rich medium unless it acquires compensatory mutations. Previously identified mutations were in<jats:italic>opuD</jats:italic>, encoding the main glycine-betaine transporter, and<jats:italic>alsT</jats:italic>, encoding a predicted amino acid transporter. Here, we show that inactivation of OpuD restores the cell size of a<jats:italic>dacA</jats:italic>mutant to near wild-type size, while inactivation of AlsT does not, suggesting two different mechanisms for the growth rescue. AlsT was identified as an efficient glutamine transporter, indicating that preventing glutamine uptake in rich medium rescues the growth of the<jats:italic>S. aureus dacA</jats:italic>mutant. In addition, GltS was identified as a glutamine transporter. By performing growth curves with WT,<jats:italic>alsT</jats:italic>and<jats:italic>gltS</jats:italic>mutant strains in defined medium supplemented with ammonium, glutamine or glutamate, we revealed that ammonium and glutamine, but not glutamate promote the growth of<jats:italic>S. aureus</jats:italic>. This suggests that besides ammonium also glutamine can serve as a nitrogen source under these conditions. Ammonium and uptake of glutamine via AlsT inhibited c-di-AMP production, while glutamate uptake had no effect. These findings provide, besides the previously reported link between potassium and osmolyte uptake, a connection between nitrogen metabolism and c-di-AMP signalling in<jats:italic>S. aureus</jats:italic>.</jats:p><jats:sec><jats:title>Graphical abstract</jats:title><jats:fig id="ufig1" position="float" fig-type="figure" orientation="portrait"><j

Journal article

Rismondo J, Halbedel S, Grundling A, 2019, Cell shape and antibiotic resistance is maintained by the activity of multiple FtsW and RodA enzymes in Listeria monocytogenes, mBio, Vol: 10, Pages: 1-17, ISSN: 2150-7511

Rod-shaped bacteria have two modes of peptidoglycan synthesis: lateral synthesis and synthesis at the cell division site. These two processes are controlled by two macromolecular protein complexes, the elongasome and divisome. Recently, it has been shown that the Bacillus subtilis RodA protein, which forms part of the elongasome, has peptidoglycan glycosyltransferase activity. The cell division specific RodA homolog FtsW fulfils a similar role at the divisome. The human pathogen Listeria monocytogenes encodes up to six FtsW/RodA homologs, however their functions have not yet been investigated. Analysis of deletion and depletion strains led to the identification of the essential cell division-specific FtsW protein, FtsW1. Interestingly, L. monocytogenes encodes a second FtsW protein, FtsW2, which can compensate for the lack of FtsW1, when expressed from an inducible promoter. L. monocytogenes also possesses three RodA homologs, RodA1, RodA2 and RodA3 and their combined absence is lethal. Cells of a rodA1/rodA3 double mutant are shorter and have increased antibiotic and lysozyme sensitivity, probably due to a weakened cell wall. Results from promoter activity assays revealed that expression of rodA3 and ftsW2 is induced in the presence of antibiotics targeting penicillin binding proteins. Consistent with this, a rodA3 mutant was more susceptible to the β-lactam antibiotic cefuroxime. Interestingly, overexpression of RodA3 also led to increased cefuroxime sensitivity. Our study highlights that L. monocytogenes encodes a multitude of functional FtsW and RodA enzymes to produce its rigid cell wall and that their expression needs to be tightly regulated to maintain growth, cell division and antibiotic resistance.

Journal article

Schuster CF, Wiedemann DM, Kirsebom FCM, Santiago M, Walker S, Gründling Aet al., 2019, High-throughput transposon sequencing highlights the cell wall as an important barrier for osmotic stress in methicillin resistantStaphylococcus aureusand underlines a tailored response to different osmotic stressors

<jats:title>Summary</jats:title><jats:p><jats:italic>Staphylococcus aureus</jats:italic>is an opportunistic pathogen that can cause soft tissue infections but is also a frequent cause of foodborne illnesses. One contributing factor for this food association is its high salt tolerance allowing this organism to survive commonly used food preservation methods. How this resistance is mediated is poorly understood, particularly during long-term exposure. In this study, we used TN-seq to understand how the responses to osmotic stressors differ. Our results revealed distinctly different long-term responses to NaCl, KCl and sucrose stresses. In addition, we identified the DUF2538 domain containing gene<jats:italic>SAUSA300_0957</jats:italic>(gene<jats:italic>957</jats:italic>) as essential under salt stress. Interestingly, a<jats:italic>957</jats:italic>mutant was less susceptible to oxacillin and showed increased peptidoglycan crosslinking. The salt sensitivity phenotype could be suppressed by amino acid substitutions in the transglycosylase domain of the penicillin binding protein Pbp2, and these changes restored the peptidoglycan crosslinking to WT levels. These results indicate that increased crosslinking of the peptidoglycan polymer can be detrimental and highlight a critical role of the bacterial cell wall for osmotic stress resistance. This study will serve as a starting point for future research on osmotic stress response and help develop better strategies to tackle foodborne staphylococcal infections.</jats:p>

Journal article

Schuster C, Howard S, Grundling A, 2019, Use of the counter selectable marker PheS* for genome engineering in Staphylococcus aureus, Microbiology, Vol: 165, Pages: 572-584, ISSN: 1350-0872

The gold standard tocreate gene deletions in Staphylococcus aureusis by homologous 16recombination using allelic exchange plasmids with a temperature sensitive origin of replication. A knockout vectorthat containsregions of homologyis first integrated into thechromosome of S. aureus by a single crossover event selected for at high temperatures (non-permissive for plasmid replication) and antibiotic selection. Next, the second crossover event is encouraged by 20growth without antibiotic selection at lowtemperature, leading at a certain frequencyto the excision of the plasmid and deletionof the gene of interest.To detector encourage plasmid loss, either a beta-galactosidase screening method or more typically, a counter selection step is used. We here present the adaption of the counter selectable markerpheS*, coding for a mutated subunit of the phenylalanine-tRNA-synthetase for use in S. aureus. The PheS* protein variant allows for the incorporation of the toxic phenylalanine amino acid analoguepara-chlorophenylalanine(PCPA)into proteins and the addition of 20-40 m M PCPAto rich medialeads to a drasticgrowth reductionof S. aureus and supplementing chemically defined medium with 2.5-5 mM PCPA to a complete growth inhibition. Using the new allelic exchange plasmid pIMAY*, wedeletedthe magnesium transporter gene mgtEin S. aureusUSA300 LAC*(SAUSA300_0910/ SAUSA300_RS04895) and RN4220(SAOUHSC_00945) and demonstrate that cobalt toxicity in S. aureusis mainly mediated by the presence of MgtE.This new plasmid will aidto efficiently and easily create gene knockouts in S. aureus.

Journal article

Rismondo J, Halbedel S, Gründling A, 2019, Cell shape and antibiotic resistance is maintained by the activity of multiple FtsW and RodA enzymes inListeria monocytogenes, Publisher: Cold Spring Harbor Laboratory

<jats:title>Abstract</jats:title><jats:p>Rod-shaped bacteria have two modes of peptidoglycan synthesis: lateral synthesis and synthesis at the cell division site. These two processes are controlled by two macromolecular protein complexes, the elongasome and divisome. Recently, it has been shown that the<jats:italic>Bacillus subtilis</jats:italic>RodA protein, which forms part of the elongasome, has peptidoglycan glycosyltransferase activity. The cell division specific RodA homolog FtsW fulfils a similar role at the divisome. The human pathogen<jats:italic>Listeria monocytogenes</jats:italic>encodes up to six FtsW/RodA homologs, however their functions have not yet been investigated. Analysis of deletion and depletion strains led to the identification of the essential cell division-specific FtsW protein, FtsW1. Interestingly,<jats:italic>L. monocytogenes</jats:italic>encodes a second FtsW protein, FtsW2, which can compensate for the lack of FtsW1, when expressed from an inducible promoter.<jats:italic>L. monocytogenes</jats:italic>also possesses three RodA homologs, RodA1, RodA2 and RodA3 and their combined absence is lethal. Cells of a<jats:italic>rodA1/rodA3</jats:italic>double mutant are shorter and have increased antibiotic and lysozyme sensitivity, probably due to a weakened cell wall. Results from promoter activity assays revealed that expression of<jats:italic>rodA3</jats:italic>and<jats:italic>ftsW2</jats:italic>is induced in the presence of antibiotics targeting penicillin binding proteins. Consistent with this, a<jats:italic>rodA3</jats:italic>mutant was more susceptible to the β-lactam antibiotic cefuroxime. Interestingly, overexpression of RodA3 also led to increased cefuroxime sensitivity. Our study highlights that<jats:italic>L. monocytogenes</jats:italic>encodes a multitude of functional FtsW and RodA enzymes to produce its

Working paper

Tosi T, Hoshiga F, Millership C, Singh R, Eldrid C, Patin D, Mengin-Lecreulx D, Thalassinos K, Freemont P, Grundling Aet al., 2019, Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM, PLoS Pathogens, Vol: 15, Pages: 1-28, ISSN: 1553-7366

c-di-AMP is an important second messenger molecule that plays a pivotal role in regulating fundamental cellular processes, including osmotic and cell wall homeostasis in many Gram-positive organisms. In the opportunistic human pathogen Staphylococcus aureus, c-di-AMP is produced by the membrane-anchored DacA enzyme. Inactivation of this enzyme leads to a growth arrest under standard laboratory growth conditions and a re-sensitization of methicillin-resistant S. aureus (MRSA) strains to ß-lactam antibiotics. The gene coding for DacA is part of the conserved three-gene dacA/ybbR/glmM operon that also encodes the proposed DacA regulator YbbR and the essential phosphoglucosamine mutase GlmM, which is required for the production of glucosamine-1-phosphate, an early intermediate of peptidoglycan synthesis. These three proteins are thought to form a complex in vivo and, in this manner, help to fine-tune the cellular c-di-AMP levels. To further characterize this important regulatory complex, we conducted a comprehensive structural and functional analysis of the S. aureus DacA and GlmM enzymes by determining the structures of the S. aureus GlmM enzyme and the catalytic domain of DacA. Both proteins were found to be dimers in solution as well as in the crystal structures. Further site-directed mutagenesis, structural and enzymatic studies showed that multiple DacA dimers need to interact for enzymatic activity. We also show that DacA and GlmM form a stable complex in vitro and that S. aureus GlmM, but not Escherichia coli or Pseudomonas aeruginosa GlmM, acts as a strong inhibitor of DacA function without the requirement of any additional cellular factor. Based on Small Angle X-ray Scattering (SAXS) data, a model of the complex revealed that GlmM likely inhibits DacA by masking the active site of the cyclase and preventing higher oligomer formation. Together these results provide an important mechanistic insight into how c-di-AMP production can be regulated in the cell.

Journal article

Karinou E, Schuster C, Pazos M, Vollmer W, Grundling Aet al., 2018, Inactivation of the monofunctional peptidoglycan glycosyltransferase SgtB allows Staphylococcus aureus to survive in the absence of lipoteichoic acid, Journal of Bacteriology, Vol: 201, ISSN: 0021-9193

The cell wall of Staphylococcus aureus is composed of peptidoglycan and the anionic polymers lipoteichoic acid (LTA) and wall teichoic acid. LTA is required for growth and normal cell morphology in S. aureus. Strains lacking LTA are usually viable only when grown under osmotically stabilizing conditions or after the acquisition of compensatory mutations. LTA-negative suppressor strains with inactivating mutations in gdpP, which resulted in increased intracellular c-di-AMP levels, were described previously. Here, we sought to identify factors other than c-di-AMP that allow S. aureus to survive without LTA. LTA-negative strains able to grow in unsupplemented medium were obtained and found to contain mutations in sgtB, mazE, clpX, or vraT. The growth improvement through mutations in mazE and sgtB was confirmed by complementation analysis. We also showed that an S. aureus sgtB transposon mutant, with the monofunctional peptidoglycan glycosyltransferase SgtB inactivated, displayed a 4-fold increase in the MIC of oxacillin, suggesting that alterations in the peptidoglycan structure could help bacteria compensate for the lack of LTA. Muropeptide analysis of peptidoglycans isolated from a wild-type strain and sgtB mutant strain did not reveal any sizable alterations in the peptidoglycan structure. In contrast, the peptidoglycan isolated from an LTA-negative ltaS mutant strain showed a significant reduction in the fraction of highly cross-linked peptidoglycan, which was partially rescued in the sgtB ltaS double mutant suppressor strain. Taken together, these data point toward an important function of LTA in cell wall integrity through its necessity for proper peptidoglycan assembly.

Journal article

Pompeo F, Rismondo J, Gründling A, Galinier Aet al., 2018, Investigation of the phosphorylation of Bacillus subtilis LTA synthases by the serine/threonine kinase PrkC, Scientific Reports, Vol: 8, ISSN: 2045-2322

Bacillus subtilis possesses four lipoteichoic acid synthases LtaS, YfnI, YvgJ and YqgS involved in the synthesis of cell wall. The crystal structure of the extracellular domain of LtaS revealed a phosphorylated threonine and YfnI was identified in two independent phosphoproteome studies. Here, we show that the four LTA synthases can be phosphorylated in vitro by the Ser/Thr kinase PrkC. Phosphorylation neither affects the export/release of YfnI nor its substrate binding. However, we observed that a phosphomimetic form of YfnI was active whereas its phosphoablative form was inactive. The phenotypes of the strains deleted for prkC or prpC (coding for a phosphatase) are fairly similar to those of the strains producing the phosphoablative or phosphomimetic YfnI proteins. Clear evidence proving that PrkC phosphorylates YfnI in vivo is still missing but our data suggest that the activity of all LTA synthases may be regulated by phosphorylation. Nonetheless, their function is non-redundant in cell. Indeed, the deletion of either ltaS or yfnI gene could restore a normal growth and shape to a ΔyvcK mutant strain but this was not the case for yvgJ or yqgS. The synthesis of cell wall must then be highly regulated to guarantee correct morphogenesis whatever the growth conditions.

Journal article

Karinou E, Schuster CF, Pazos M, Vollmer W, Gründling Aet al., 2018, Inactivation of the monofunctional peptidoglycan glycosyltransferase SgtB allowsStaphylococcus aureusto survive in the absence of lipoteichoic acid, Publisher: Cold Spring Harbor Laboratory

<jats:title>Abstract</jats:title><jats:p>The cell wall of<jats:italic>Staphylococcus aureus</jats:italic>is composed of peptidoglycan and the anionic polymers lipoteichoic acid (LTA) and wall teichoic acid. LTA is required for growth and normal cell morphology in<jats:italic>S. aureus.</jats:italic>Strains lacking LTA are usually only viable when grown under osmotically stabilizing conditions or after the acquisition of compensatory mutations. LTA negative suppressor strains with inactivating mutations in<jats:italic>gdpP</jats:italic>, resulting in an increase in intracellular c-di-AMP levels, have been described previously. Here, we sought to identify factors other than c-di-AMP that allow<jats:italic>S. aureus</jats:italic>to survive without LTA. LTA-negative strains able to grow in un-supplemented medium were obtained and found to contain mutations in<jats:italic>sgtB, mazE, clpX</jats:italic>or<jats:italic>vraT</jats:italic>. The growth improvement through mutations in<jats:italic>mazE</jats:italic>and<jats:italic>sgtB</jats:italic>was confirmed by complementation analysis. We also show that an<jats:italic>S. aureus sgtB</jats:italic>transposon mutant, inactivated for the monofunctional peptidoglycan glycosyltransferase SgtB, displays a 4-fold increase in the MIC towards a number of cell wall-targeting antibiotics, suggesting that alteration in the peptidoglycan structure could help bacteria compensate for the lack of LTA. Muropeptide analysis of peptidoglycan isolated from a WT and<jats:italic>sgtB</jats:italic>mutant strains did not reveal any sizable alternations in the peptidoglycan structure. In contrast, the peptidoglycan isolated from an LTA-negative<jats:italic>ltaS</jats:italic>mutant strain showed a significant reduction in the fraction of highly crosslinked peptidoglycan, which was partially rescued in

Working paper

Rismondo J, Percy MG, Gründling A, 2018, Discovery of genes required for lipoteichoic acid glycosylation predicts two distinct mechanisms for wall teichoic acid glycosylation., J Biol Chem, Vol: 293, Pages: 3293-3306

The bacterial cell wall is an important and highly complex structure that is essential for bacterial growth because it protects bacteria from cell lysis and environmental insults. A typical Gram-positive bacterial cell wall is composed of peptidoglycan and the secondary cell wall polymers, wall teichoic acid (WTA) and lipoteichoic acid (LTA). In many Gram-positive bacteria, LTA is a polyglycerol-phosphate chain that is decorated with d-alanine and sugar residues. However, the function of and proteins responsible for the glycosylation of LTA are either unknown or not well-characterized. Here, using bioinformatics, genetic, and NMR spectroscopy approaches, we found that the Bacillus subtilis csbB and yfhO genes are essential for LTA glycosylation. Interestingly, the Listeria monocytogenes gene lmo1079, which encodes a YfhO homolog, was not required for LTA glycosylation, but instead was essential for WTA glycosylation. LTA is polymerized on the outside of the cell and hence can only be glycosylated extracellularly. Based on the similarity of the genes coding for YfhO homologs that are required in B. subtilis for LTA glycosylation or in L. monocytogenes for WTA glycosylation, we hypothesize that WTA glycosylation might also occur extracellularly in Listeria species. Finally, we discovered that in L. monocytogenes, lmo0626 (gtlB) was required for LTA glycosylation, indicating that the encoded protein has a function similar to that of YfhO, although the proteins are not homologous. Together, our results enable us to propose an updated model for LTA glycosylation and also indicate that glycosylation of WTA might occur through two different mechanisms in Gram-positive bacteria.

Journal article

Zeden MS, Schuster CF, Bowman L, Zhong Q, Williams HD, Grundling Aet al., 2018, Cyclic-di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation in Staphylococcus aureus but dispensable for viability in anaerobic conditions, Journal of Biological Chemistry, Vol: 293, Pages: 3180-3200, ISSN: 0021-9258

Cyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered signaling molecule important for the survival of Firmicutes, a large bacterial group that includes notable pathogens such as Staphylococcus aureus. However, the exact role of this molecule has not been identified. dacA, the S. aureus gene encoding the diadenylate cyclase enzyme required for c-di-AMP production, cannot be deleted when bacterial cells are grown in rich medium, indicating that c-di-AMP is required for growth in this condition. Here, we report that an S. aureus dacA mutant can be generated in chemically defined medium. Consistent with previous findings, this mutant had a severe growth defect when cultured in rich medium. Using this growth defect in rich medium, we selected for suppressor strains with improved growth to identify c-di-AMP-requiring pathways. Mutations bypassing the essentiality of dacA were identified in alsT and opuD, encoding a predicted amino acid and osmolyte transporter, the latter of which we show here to be the main glycine betaine-uptake system in S. aureus. Inactivation of these transporters likely prevents the excessive osmolyte and amino acid accumulation in the cell, providing further evidence for a key role of c-di-AMP in osmotic regulation. Suppressor mutations were also obtained in hepS, hemB, ctaA and qoxB, coding for proteins required for respiration. Furthermore, we show that dacA is dispensable for growth in anaerobic conditions. Together, these finding reveal an essential role for the c-di-AMP signaling network in aerobic, but not anaerobic, respiration in S. aureus.

Journal article

Zeden MS, Schuster CF, Bowman L, Zhong Q, Williams HD, Gründling Aet al., 2017, Cyclic-di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation inStaphylococcus aureusbut dispensable for viability in anaerobic conditions, Publisher: Cold Spring Harbor Laboratory

<jats:title>ABSTRACT</jats:title><jats:p>Cyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered signaling molecule important for the survival of Firmicutes, a large bacterial group that includes notable pathogens such as<jats:italic>Staphylococcus aureus</jats:italic>. However, the exact role of this molecule has not been identified.<jats:italic>dacA</jats:italic>, the<jats:italic>S. aureus</jats:italic>gene encoding the diadenylate cyclase enzyme required for c-di-AMP production, cannot be deleted when bacterial cells are grown in rich medium, indicating that c-di-AMP is required for growth in this condition. Here, we report that an<jats:italic>S. aureus dacA</jats:italic>mutant can be generated in chemically defined medium. Consistent with previous findings, this mutant had a severe growth defect when cultured in rich medium. Using this growth defect in rich medium, we selected for suppressor strains with improved growth to identify c-di-AMP-requiring pathways. Mutations bypassing the essentiality of<jats:italic>dacA</jats:italic>were identified in<jats:italic>alsT</jats:italic>and<jats:italic>opuD</jats:italic>, encoding a predicted amino acid and osmolyte transporter, the latter of which we show here to be the main glycine betaine-uptake system in<jats:italic>S. aureus</jats:italic>. Inactivation of these transporters likely prevents the excessive osmolyte and amino acid accumulation in the cell, providing further evidence for a key role of c-di-AMP in osmotic regulation. Suppressor mutations were also obtained in<jats:italic>hepS, hemB, ctaA</jats:italic>and<jats:italic>qoxB</jats:italic>, coding for proteins required for respiration. Furthermore, we show that<jats:italic>dacA</jats:italic>is dispensable for growth in anaerobic conditions. Together, these finding reveal an essential role for the c-d

Working paper

Zhang Y, Agrebi R, Bellows LE, Collet JF, Kaever V, Gründling Aet al., 2016, Evolutionary adaptation of the essential tRNA methyltransferase TrmD to the signaling molecule 3,5-cAMP in bacteria., Journal of Biological Chemistry, Vol: 292, Pages: 313-327, ISSN: 1083-351X

The nucleotide signaling molecule 3',5'-cyclic adenosine monophosphate (3',5'-cAMP) plays important physiological roles, ranging from carbon catabolite repression in bacteria to mediating the action of hormones in higher eukaryotes, including human. However, it remains unclear whether 3',5'-cAMP is universally present in the Firmicutes group of bacteria. We hypothesized that searching for proteins that bind 3',5'-cAMP might provide new insight into this question. Accordingly, we performed a genome-wide screen, and identified the essential Staphylococcus aureus tRNA m1G37 methyltransferase enzyme TrmD, which is conserved in all three domains of life, as a tight 3',5'-cAMP binding protein. TrmD enzymes are known to use S-adenosyl-L-methionine (AdoMet) as substrate; we shown that 3',5'-cAMP binds competitively with AdoMet to the S. aureus TrmD protein, indicating an overlapping binding site. However, the physiological relevance of this discovery remained unclear, as we were unable to identify a functional adenylate cyclase in S. aureus and only detected 2',3'-cAMP but not 3',5'-cAMP in cellular extracts. Interestingly, TrmD proteins from Escherichia coli and Mycobacterium tuberculosis, organisms known to synthesize 3',5'-cAMP, did not bind this signaling nucleotide. Comparative bioinformatics, mutagenesis and biochemical analyses revealed that the highly conserved Tyr86 residue in E. coli TrmD is essential to discriminate between 3',5'-cAMP and the native substrate AdoMet. Combined with a phylogenetic analysis, these results suggest that amino acids in the substrate binding pocket of TrmD underwent an adaptive evolution to accommodate the emergence of adenylate cyclases and thus the signaling molecule 3',5'-cAMP. Altogether this further indicates that S. aureus does not produce 3',5'-cAMP, which would otherwise competitively inhibit an essential enzyme.

Journal article

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