Imperial College London

ProfessorAngelikaGrundling

Faculty of MedicineDepartment of Medicine

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

56 results found

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, ISSN: 2150-7511

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 in Listeria 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 enzy

Working paper

Schuster C, Howard S, Grundling A, 2019, Use of the counter selectable marker PheS* for genome engineering in Staphylococcus aureus, Microbiology, 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

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, 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, Grundling Aet al., 2018, Inactivation of the monofunctional peptidoglycan glycosyltransferase SgtB allows Staphylococcus aureus to survive in the absence of lipoteichoic acid, Publisher: Cold Spring Harbor Laboratory

<jats:p>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 only viable when grown under osmotically stabilizing conditions or after the acquisition of compensatory mutations. LTA negative suppressor strains with inactivating mutations in gdpP, 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 S. aureus to survive without LTA. LTA-negative strains able to grow in un-supplemented 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 show that an S. aureus sgtB 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 sgtB mutant strains did not reveal any sizable alternations 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 crosslinked peptidoglycan, which was partially rescued in the sgtB/ltaS double mutant suppressor strain. Taken together, these data point towards an important function of LTA in cell wall integrity through its requirement for proper peptidoglycan assembly.</jats:p>

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, Grundling Aet al., 2017, Cyclic-di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation in Staphylococcus aureus but dispensable for viability in anaerobic conditions, Publisher: Cold Spring Harbor Laboratory

<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 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.</jats:p>

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

Bowman L, Zeden MS, Schuster CF, Kaever V, Grundling Aet al., 2016, New Insights into the Cyclic di-Adenosine Monophosphate (c-di-AMP) Degradation Pathway and the Requirement of the Cyclic-Dinucleotide for Acid Stress Resistance in Staphylococcus aureus., Journal of Biological Chemistry, Vol: 291, Pages: 26970-26986, ISSN: 1083-351X

Nucleotide signaling networks are key to facilitate alterations in gene expression, protein function and enzyme activity in response to diverse stimuli. Cyclic di-adenosine monophosphate (c-di-AMP) is an important secondary messenger molecule produced by the human pathogen Staphylococcus aureus and is involved in regulating a number of physiological processes including potassium transport. S. aureus must ensure tight control over its cellular levels as both high levels of the dinucleotide and its absence result in a number of detrimental phenotypes. Here we show that in addition to the membrane bound Asp-His-His and Asp-His-His associated (DHH/DHHA1) domain-containing phosphodiesterase (PDE) GdpP, S. aureus produces a second cytoplasmic DHH/DHHA1 PDE Pde2. Although capable of hydrolyzing c-di-AMP, Pde2 preferentially converts linear 5-phosphadenylyl-adenosine (pApA) to AMP. Using a pde2 mutant strain, pApA was detected for the first time in S. aureus, leading us to speculate that this dinucleotide may have a regulatory role under certain conditions. Moreover, pApA is involved in a feedback inhibition loop that limits GdpP-dependent c-di-AMP hydrolysis. Another protein linked to the regulation of c-di-AMP levels in bacteria is the predicted regulator protein YbbR. Here, it is shown that a ybbR mutant S. aureus strain has increased acid sensitivity that can be bypassed by the acquisition of mutations in a number of genes, including the gene coding for the diadenylate cyclase DacA. We further show that c-di-AMP levels are slightly elevated in the ybbR suppressor strains tested as compared to the wild-type strain. With this, we not only identified a new role for YbbR in acid stress resistance in S. aureus, but also provide further insight into how c-di-AMP levels impact acid tolerance in this organism.

Journal article

Grundling A, Lee V, 2016, Old concepts, new molecules and current approaches applied to the bacterial nucleotide signalling field, Philosophical Transactions of the Royal Society B: Biological Sciences, Vol: 371, ISSN: 1471-2970

Signalling nucleotides are key molecules that help bacteria to rapidly coordinate cellular pathways and adaptto changes in their environment. During the past ten years, the nucleotide-signalling field has seen muchexcitement, as several new signalling nucleotides have been discovered in both eukaryotic and bacterial cells.The fields have since advanced quickly, aided by the development of important tools such as the synthesis ofmodified nucleotides, which combined with sensitive mass spectrometry methods, allowed for the rapididentification of specific receptor proteins along with other novel genome-wide screening methods. In thisreview, we will describe the principle concepts of nucleotide signalling networks and summarize the recentwork that led to the discovery of the novel signalling nucleotides. We will also highlight current approachesapplied to the research in the field as well as resources and methodological advances aiding in a rapididentification of nucleotide specific receptor proteins.

Journal article

Schuster C, Bellows L, Tosi T, Campeotto, Corrigan, Freemont P, Grundling Aet al., 2016, The second messenger c-di-AMP inhibits the osmolyte uptake system OpuC in Staphylococcus aureus, Science Signaling, Vol: 9, Pages: ra81-ra81, ISSN: 1945-0877

Staphylococcus aureus is an important opportunistic human pathogen that is highly resistant to osmotic stresses. To survive an increase in osmolarity, bacteria immediately take up potassium ions and small organic compounds known as compatible solutes. The second messenger cyclic diadenosine monophosphate (c-di-AMP) reduces the ability of bacteria to withstand osmotic stress by binding to and inhibiting several proteins that promote potassium uptake. We identified OpuCA, the adenosine triphosphatase (ATPase) component of an uptake system for the compatible solute carnitine, as a c-di-AMP target protein in S. aureus and found that the LAC*ΔgdpP strain of S. aureus, which overproduces c-di-AMP, showed reduced carnitine uptake. The paired cystathionine-β-synthase (CBS) domains of OpuCA bound to c-di-AMP, and a crystal structure revealed a putative binding pocket for c-di-AMP in the cleft between the two CBS domains. Thus, c-di-AMP inhibits osmoprotection through multiple mechanisms.

Journal article

Baek K, Bowman L, Millership C, Dupont Sogaard M, Kaever V, Savijoki K, Varmanen P, Nyman T, Grundling A, Frees Det al., 2016, The cell wall polymer lipoteichoic acid becomes non-essential in Staphylococcus aureus cells lacking the ClpX chaperone, mBio, Vol: 7, ISSN: 2150-7511

Lipoteichoic acid is an essential component of the Staphylococcus aureus cell envelope and an attractive target for the development of vaccines and antimicrobials directed against antibiotic-resistant Gram-positive bacteria such as methicillin-resistant S. aureus and vancomycin-resistant enterococci. In this study, we showed that the lipoteichoic acid polymer is essential for growth of S. aureus only as long as the ClpX chaperone is present in the cell. Our results indicate that lipoteichoic acid and ClpX play opposite roles in a pathway that controls two key cell division processes in S. aureus, namely, septum formation and autolytic activity. The discovery of a novel functional connection in the genetic network that controls cell division in S. aureus may expand the repertoire of possible strategies to identify compounds or compound combinations that kill antibiotic-resistant S. aureus.

Journal article

Percy M, Karinou E, Webb A, Grundling Aet al., 2016, Identification of a lipoteichoic acid glycosyltransferase enzyme reveals that GW-domain containing proteins can be retained in the cell wall of Listeria monocytogenes in the absence of lipoteichoic acid or its modifications, Journal of Bacteriology, Vol: 198, Pages: 2029-2042, ISSN: 1098-5530

Listeria monocytogenes is a food-borne Gram-positive bacterial pathogen and many of its virulence factors are either secreted proteins, or proteins covalently or non-covalently-attached to the cell wall. Previous work has indicated that non-covalently-attached proteins with GW domains are retained in the cell wall by binding to the cell wall polymer lipoteichoic acid (LTA). LTA is a glycerolphosphate polymer, which is modified in L. monocytogenes with galactose and D-alanine residues. We identified Lmo0933 as the cytoplasmic glycosyltransferase required for the LTA glycosylation process and renamed the protein GtlA for glycosyltransferase LTA A. Using L. monocytogenes mutants lacking galactose or D-alanine modifications or the complete LTA polymer, we show that GW-domain proteins are still retained within the cell wall, indicating that other cell wall polymers are involved in the retention of GW-domain proteins. Further experiments reveal peptidoglycan as the binding receptor as a purified GW domainfusion protein can bind to L. monocytogenes cells lacking wall teichoic acid (WTA) as well as purified peptidoglycan derived from a wild-type or WTA-negative strain. With this, we not only identified the first enzyme involved in the LTA glycosylation process, but we also provide new insight into the binding mechanism of non-covalently attached cell wall proteins.

Journal article

Corrigan RM, Bellows LE, Wood A, Grundling Aet al., 2016, ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria, Proceedings of the National Academy of Sciences of the United States of America, Vol: 113, Pages: E1710-E1719, ISSN: 1091-6490

The stringent response is a survival mechanism used by bacteria to deal with stress. It is coordinated by the nucleotides guanosine tetraphosphate and pentaphosphate [(p)ppGpp], which interact with target proteins to promote bacterial survival. Although this response has been well characterized in proteobacteria, very little is known about the effectors of this signaling system in Gram-positive species. Here, we report on the identification of seven target proteins for the stringent response nucleotides in the Gram-positive bacterium Staphylococcus aureus. We demonstrate that the GTP synthesis enzymes HprT and Gmk bind with a high affinity, leading to an inhibition of GTP production. In addition, we identified five putative GTPases—RsgA, RbgA, Era, HflX, and ObgE—as (p)ppGpp target proteins. We show that RsgA, RbgA, Era, and HflX are functional GTPases and that their activity is promoted in the presence of ribosomes but strongly inhibited by the stringent response nucleotides. By characterizing the function of RsgA in vivo, we ascertain that this protein is involved in ribosome assembly, with an rsgA deletion strain, or a strain inactivated for GTPase activity, displaying decreased growth, a decrease in the amount of mature 70S ribosomes, and an increased level of tolerance to antimicrobials. We additionally demonstrate that the interaction of ppGpp with cellular GTPases is not unique to the staphylococci, as homologs from Bacillus subtilis and Enterococcus faecalis retain this ability. Taken together, this study reveals ribosome inactivation as a previously unidentified mechanism through which the stringent response functions in Gram-positive bacteria.

Journal article

Moscoso JA, Schramke H, Zhang Y, Tosi T, Dehbi A, Jung K, Gründling Aet al., 2015, Binding of cyclic Di-AMP to the staphylococcus aureus sensor kinase KdpD occurs via the universal stress protein domain and downregulates the expression of the Kdp potassium transporter, Journal of Bacteriology, ISSN: 1098-5530

Nucleotide signalling molecules are important intracellular messengers that regulate a wide range of biological functions. The human pathogen Staphylococcus aureus produces the signalling nucleotide cyclic di-adenosine monophosphate (c-di-AMP). This molecule is common among Gram-positive bacteria and in many organisms essential for survival under standard laboratory growth conditions. In this study, we investigated the interaction of c-di-AMP with the S. aureus KdpD protein. The sensor kinase KdpD forms a two-component signalling system with the response regulator KdpE and regulates the expression of the kdpDE genes and the kdpFABC operon coding for the Kdp potassium transporter components. Here, we show that the S. aureus KdpD protein binds c-di-AMP specifically and with an affinity in the micromolar range through its universal stress protein (USP) domain. This domain is located within the N-terminal cytoplasmic region of KdpD and amino acids of a conserved SxS-X20-FTAxY motif are important for this binding. We further show that KdpD2, a second KdpD protein found in some S. aureus strains, also binds c-di-AMP and our bioinformatics analysis indicates that a subclass of KdpD proteins in c-di-AMP-producing bacteria has evolved to bind this signalling nucleotide. Finally, we show that c-di-AMP binding to KdpD inhibits the up-regulation of the kdpFABC operon under salt stress, thus indicating that c-di-AMP is a negative regulator of potassium uptake in S. aureus. IMPORTANCE: Staphylococcus aureus is an important human pathogen and major cause of food poisoning in western countries. A common method for food preservation is the use of salt to drive dehydration. This study sheds light on the regulation of potassium uptake in Staphylococcus aureus, an important aspect of this bacterium's ability to tolerate high levels of salt. We show that the signalling nucleotide c-di-AMP binds to a regulatory component of the Kdp potassium uptake system and that this binding has an inh

Journal article

Hengge R, Gruendling A, Jenal U, Ryan R, Yildiz Fet al., 2015, Bacterial signal transduction by cyclic Di-GMP and other nucleotide second messengers, Journal of Bacteriology, Vol: 198, Pages: 15-26, ISSN: 1098-5530

The first International Symposium on c-Di-GMP Signaling in Bacteria (22 to 25 March 2015, Harnack-Haus, Berlin, Germany) brought together 131 molecular microbiologists from 17 countries to discuss recent progress in our knowledge of bacterial nucleotide second messenger signaling. While the focus was on signal input, synthesis, degradation, and the striking diversity of the modes of action of the current second messenger paradigm, i.e., cyclic di-GMP (c-di-GMP), “classics” like cAMP and (p)ppGpp were also presented, in novel facets, and more recent “newcomers,” such as c-di-AMP and c-AMP-GMP, made an impressive appearance. A number of clear trends emerged during the 30 talks, on the 71 posters, and in the lively discussions, including (i) c-di-GMP control of the activities of various ATPases and phosphorylation cascades, (ii) extensive cross talk between c-di-GMP and other nucleotide second messenger signaling pathways, and (iii) a stunning number of novel effectors for nucleotide second messengers that surprisingly include some long-known master regulators of developmental pathways. Overall, the conference made it amply clear that second messenger signaling is currently one of the most dynamic fields within molecular microbiology, with major impacts in research fields ranging from human health to microbial ecology.

Journal article

Gross CA, Gruendling A, 2015, Editorial overview: Cell regulation: When you think you know it all, there is another layer to be discovered, CURRENT OPINION IN MICROBIOLOGY, Vol: 24, Pages: V-VII, ISSN: 1369-5274

Journal article

Corrigan RM, Bowman L, Willis AR, Kaever V, Gruendling Aet al., 2015, Cross-talk between Two Nucleotide-signaling Pathways in Staphylococcus aureus, Journal of Biological Chemistry, Vol: 290, Pages: 5826-5839, ISSN: 0021-9258

Nucleotide-signaling pathways are found in all kingdoms oflife and are utilized to coordinate a rapid response to externalstimuli. The stringent response alarmones guanosine tetra(ppGpp)and pentaphosphate (pppGpp) control a globalresponse allowing cells to adapt to starvation conditions such asamino acid depletion. One more recently discovered signalingnucleotide is the secondary messenger cyclic diadenosinemonophosphate (c-di-AMP). Here, we demonstrate that thissignaling nucleotide is essential for the growth of Staphylococcusaureus, and its increased production during late growthphases indicates that c-di-AMP controls processes that areimportant for the survival of cells in stationary phase. By examiningthe transcriptional profile of cells with high levels of c-diAMP,we reveal a significant overlap with a stringent responsetranscription signature. Examination of the intracellular nucleotidelevels under stress conditions provides further evidencethat high levels of c-di-AMP lead to an activation of the stringentresponse through a RelA/SpoT homologue (RSH) enzymedependentincrease in the (p)ppGpp levels. This activation isshown to be indirect as c-di-AMP does not interact directly withthe RSH protein. Our data extend this interconnection furtherby showing that the S. aureus c-di-AMP phosphodiesteraseenzyme GdpP is inhibited in a dose-dependent manner byppGpp, which itself is not a substrate for this enzyme. Altogether,these findings add a new layer of complexity to ourunderstanding of nucleotide signaling in bacteria as they highlightintricate interconnections between different nucleotidesignalingnetworks.

Journal article

Campeotto I, Zhang Y, Mladenov MG, Freemont PS, Grundling Aet al., 2015, Complex Structure and Biochemical Characterization of the Staphylococcus aureus Cyclic Diadenylate Monophosphate (c-di-AMP)-binding Protein PstA, the Founding Member of a New Signal Transduction Protein Family, Journal of Biological Chemistry, Vol: 290, Pages: 2888-2901, ISSN: 1083-351X

Signaling nucleotides are integral parts of signal transductionsystems allowing bacteria to cope with and rapidly respond tochanges in the environment. The Staphylococcus aureus PII-likesignal transduction protein PstA was recently identified as acyclic diadenylate monophosphate (c-di-AMP)-binding protein.Here, we present the crystal structures of the apo- and c-diAMP-boundPstA protein, which is trimeric in solution as wellas in the crystals. The structures combined with detailed bioinformaticsanalysis revealed that the protein belongs to a newfamily of proteins with a similar core fold but with distinct featuresto classical PII proteins, which usually function in nitrogenmetabolism pathways in bacteria. The complex structurerevealed three identical c-di-AMP-binding sites per trimer witheach binding site at a monomer-monomer interface. Althoughdistinctly different from other cyclic-di-nucleotide-bindingsites, as the half-binding sites are not symmetrical, the complexstructure also highlighted common features for c-di-AMPbindingsites. A comparison between the apo and complexstructures revealed a series of conformational changes thatresult in the ordering of two anti-parallel !-strands that protrudefrom each monomer and allowed us to propose a mechanismon how the PstA protein functions as a signaling transductionprotein.

Journal article

Campeotto I, Percy MG, MacDonald JT, Foerster A, Freemont PS, Gruendling Aet al., 2014, Structural and Mechanistic Insight into the Listeria monocytogenes Two-enzyme Lipoteichoic Acid Synthesis System, Journal of Biological Chemistry, Vol: 289, Pages: 28054-28069, ISSN: 0021-9258

Lipoteichoic acid (LTA) is an important cell wall componentrequired for proper cell growth in many Gram-positive bacteria.In Listeria monocytogenes, two enzymes are required for the synthesisof this polyglycerolphosphate polymer. The LTA primaseLtaPLm initiates LTA synthesis by transferring the first glycerolphosphate(GroP) subunit onto the glycolipid anchor and theLTA synthase LtaSLm extends the polymer by the repeated additionof GroP subunits to the tip of the growing chain. Here, wepresent the crystal structures of the enzymatic domains ofLtaPLm and LtaSLm. Although the enzymes share the same fold,substantial differences in the cavity of the catalytic site andsurface charge distribution contribute to enzyme specialization.The eLtaSLm structure was also determined in complexwith GroP revealing a second GroP binding site. Mutationalanalysis confirmed an essential function for this binding siteand allowed us to propose a model for the binding of thegrowing chain.

Journal article

Baek KT, Grundling A, Mogensen RG, Thogersen L, Petersen A, Paulander W, Frees Det al., 2014, beta-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus USA300 Is Increased by Inactivation of the ClpXP Protease, ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Vol: 58, Pages: 4593-4603, ISSN: 0066-4804

Journal article

Reichmann NT, Cassona CP, Monteiro JM, Bottomley AL, Corrigan RM, Foster SJ, Pinho MG, Gruendling Aet al., 2014, Differential localization of LTA synthesis proteins and their interaction with the cell division machinery in Staphylococcus aureus, MOLECULAR MICROBIOLOGY, Vol: 92, Pages: 273-286, ISSN: 0950-382X

Journal article

Percy MG, Gruendling A, 2014, Lipoteichoic Acid Synthesis and Function in Gram-Positive Bacteria, ANNUAL REVIEW OF MICROBIOLOGY, VOL 68, Vol: 68, Pages: 81-100, ISSN: 0066-4227

Journal article

Mercedes Palomino M, Allievi MC, Gruendling A, Sanchez-Rivas C, Ruzal SMet al., 2013, Osmotic stress adaptation in Lactobacillus casei BL23 leads to structural changes in the cell wall polymer lipoteichoic acid, MICROBIOLOGY-SGM, Vol: 159, Pages: 2416-2426, ISSN: 1350-0872

Journal article

Reichmann NT, Cassona CP, Gruendling A, 2013, Revised mechanism of D-alanine incorporation into cell wall polymers in Gram-positive bacteria, MICROBIOLOGY-SGM, Vol: 159, Pages: 1868-1877, ISSN: 1350-0872

Journal article

Gruendling A, 2013, Potassium Uptake Systems in Staphylococcus aureus: New Stories about Ancient Systems, MBIO, Vol: 4, ISSN: 2150-7511

Journal article

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