Biology subjects

Grundling, A.

Publications and source records attributed to Grundling, A..

11 recordsLinked to original sources

bioRxiv2024

Cytosolic factors controlling PASTA kinase-dependent ReoM phosphorylation

Bacteria adapt the biosynthesis of their envelopes to specific growth conditions and prevailing stress factors. Peptidoglycan (PG) is the major component of the cell wall in Gram-positive bacteria, where PASTA kinases play a central role in PG biosynthesis regulation. Despite their importance for growth, cell division and antibiotic resistance, the mechanisms of PASTA kinase activation are not fully understood. ReoM, a recently discovered cytosolic phosphoprotein, is one of the main substrates of the PASTA kinase PrkA in the Gram-positive human pathogen Listeria monocytogenes. Depending on its phosphorylation, ReoM controls proteolytic stability of MurA, the first enzyme in the PG biosynthesis pathway. The late cell division protein GpsB has been implicated in PASTA kinase signalling. Consistently, we show that L. monocytogenes prkA and gpsB mutants phenocopied each other. Analysis of in vivo ReoM phosphorylation confirmed GpsB as an activator of PrkA leading to the description of structural features in GpsB that are important for kinase activation. We further show that ReoM phosphorylation is growth-phase dependent and that this kinetic is reliant on the protein phosphatase PrpC. ReoM phosphorylation was inhibited in mutants with defects in MurA degradation, leading to the discovery that artificial MurA overexpression prevented ReoM phosphorylation. Overexpressed MurA must adopt its substrate-bound closed conformation and interact with ReoM to exert this effect, but the extracellular PASTA domains of PrkA or MurJ flippases where not required. Our results indicate that intracellular signals control ReoM phosphorylation and extend current models describing the mechanisms of PASTA kinase activation.

microbiology↗

bioRxiv2023

Histidine and its uptake are essential for the growth of Staphylococcus aureus at low pH

Staphylococcus aureus is an opportunistic pathogen capable of causing many different human diseases. During colonization and infection, S. aureus will encounter a range of hostile environments, including acidic conditions such as those found on the skin and within macrophages. However, little is known about the mechanisms that S. aureus uses to detect and respond to low pH. Here, we employed a transposon sequencing approach to determine on a genome-wide level the genes required or detrimental for growth at low pH. We identified 31 genes that were essential for the growth of S. aureus at pH 4.5 and confirmed the importance of many of them through follow up experiments using mutant strains inactivated for individual genes. Most of the genes identified code for proteins with functions in cell wall assembly and maintenance. These data suggest that the cell wall has a more important role than previously appreciated in promoting bacterial survival when under acid stress. We also identified several novel processes previously not linked to the acid stress response in S. aureus. These include aerobic respiration and histidine transport, the latter by showing that one of the most important genes, SAUSA300_0846, codes for a previously uncharacterized histidine transporter. We show that an S. aureus SAUSA300_0846 mutant is unable to maintain its cytosolic pH, thereby revealing an important function for histidine and its transport in buffering the intracellular pH in bacteria. Author summaryStaphylococcus aureus is an important human bacterial pathogen that can cause a range of diseases. During infection, the pathogen will encounter a hostile environment within the human host, including acidic conditions such as those found on the skin and within macrophages. The bacterium has developed sophisticated strategies to survive and grow under such harsh conditions. Here we performed a genome wide screen to identify factors that are required by this pathogen to survive under acid stress conditions and identified several novel processes including histidine uptake. Understanding the response of S. aureus to deal with acid stress conditions will help us better manage infections.

microbiology↗

bioRxiv2021

Complex structure of the Bacillus subtilis CdaA c-di-AMP cyclase domain and the phosphoglucomutase GlmM

Cyclic-di-adenosine monophosphate (c-di-AMP) is an important nucleotide signalling molecule, which plays a key role in osmotic regulation in bacteria. Cellular c-di-AMP levels are tightly regulated, as both high and low levels have a negative impact on bacterial growth. Here, we investigated how the activity of the main Bacillus subtilis c-di-AMP cyclase CdaA is regulated by the phosphoglucomutase GlmM. c-di-AMP is produced from two molecules of ATP by proteins containing a deadenylate cyclase (DAC) domain. CdaA is a membrane-linked cyclase with an N-terminal transmembrane domain followed by the cytoplasmic DAC domain. Here we show, using the soluble catalytic B. subtilis CdaACD domain and purified full-length GlmM or the GlmMF369 variant lacking the C-terminal flexible domain 4, that the cyclase and phosphoglucomutase form a stable complex in vitro and that GlmM is a potent cyclase inhibitor. We determined the crystal structure of the individual B. subtilis CdaACD and GlmM proteins, both of which form dimers in the structures, and of the CdaACD:GlmMF369 complex. In the complex structure, a CdaACD dimer is bound to a GlmMF369 dimer in such a manner that GlmM blocks the oligomerization of CdaACD and formation of active head-to-head cyclase oligomers, thus providing molecular details on how GlmM acts as cyclase inhibitor. The function of a key amino acid residue in CdaACD in complex formation was confirmed by mutagenesis analysis. As the amino acids at the CdaACD:GlmM interphase are conserved, we propose that the observed inhibition mechanism of CdaA by GlmM is conserved among Firmicutes.

microbiology↗

bioRxiv2020

Bacillus subtilis YngB is a UTP-glucose-1-phosphate uridylyltransferase contributing to wall teichoic acid modification and glycolipid formation during anaerobic growth

UTP-glucose-1-phosphate uridylyltransferases (UGPases) 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 UGPases, 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 wild-type 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.

microbiology↗

bioRxiv2020

Phosphoglycerol-type wall- and lipoteichoic acids are enantiomeric polymers differentially cleaved by the stereospecific glycerophosphodiesterase GlpQ

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 Bacillus subtilis 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 sn-glycerol-3-phosphodiesterase GlpQ can discriminate between B. subtilis 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 sn-glycerol-3-phosphate and LTA is made of sn-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, B. subtilis 168 specifically hydrolyzes WTA and synthesizes phosphate-free teichuronic acids in exchange.

microbiology↗

bioRxiv2019

GtcA is required for LTA glycosylation in Listeria monocytogenes serovar 1/2a and Bacillus subtilis

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. GRAPHICAL ABSTRACT O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

microbiology↗

bioRxiv2019

Staphylococcus aureus can survive in the absence of c-di-AMP upon inactivation of the main glutamine transporter

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 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 S. aureus dacA mutant. In addition, GltS was identified as a glutamine 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 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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/754309v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1f6f305org.highwire.dtl.DTLVardef@1cceafeorg.highwire.dtl.DTLVardef@57cba5org.highwire.dtl.DTLVardef@1fabed6_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2019

High-throughput transposon sequencing highlights cell wall as an important barrier for osmotic stress in methicillin resistant Staphylococcus aureus and underlines a tailored response to different osmotic stressors

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.

microbiology↗

bioRxiv2019

Balanced activity of multiple FtsW and RodA proteins is crucial for maintaining cell shape and antibiotic resistance in Listeria monocytogenes

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 {beta}-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.\n\nImportanceThe human pathogen Listeria monocytogenes is usually treated with high doses of {beta}-lactam antibiotics, often combined with gentamicin. However, these antibiotics only act bacteriostatically on L. monocytogenes and the immune system is needed to clear the infection. Therefore, individuals with a compromised immune system are at risk to develop a severe form of Listeria infection, which can be fatal in up to 30% of cases. The development of new strategies to treat Listeria infections is therefore necessary. Here we show that the expression of some of the FtsW and RodA enzymes of L. monocytogenes is induced by the presence of {beta}-lactam antibiotics and their combined absence makes bacteria more susceptible to this class of antibiotics. The development of antimicrobials that inhibit the activity or production of FtsW/RodA enzymes might therefore help to improve the treatment of Listeria infections and thereby lead to a reduction in mortality.

microbiology↗

bioRxiv2018

Inactivation of the monofunctional peptidoglycan glycosyltransferase SgtB allows Staphylococcus aureus to survive in the absence of lipoteichoic acid

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.\n\nImportanceThe bacterial cell wall acts as primary defence against environmental insults such as changes in osmolarity. It is also a vulnerable structure as defects in its synthesis can lead to growth arrest or cell death. The important human pathogen Staphylococcus aureus has a typical Gram-positive cell wall, which consists of peptidoglycan and the anionic polymers lipoteichoic acid (LTA) and wall teichoic acid. Several clinically relevant antibiotics inhibit the synthesis of peptidoglycan; hence it and teichoic acids are considered attractive targets for the development of new antimicrobials. We show that LTA is required for efficient peptidoglycan crosslinking in S. aureus and inactivation of a peptidoglycan glycosyltransferase can partially rescue this defect, altogether revealing an intimate link between peptidoglycan and LTA synthesis.

microbiology↗

bioRxiv2017

Cyclic-di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation in Staphylococcus aureus but dispensable for viability in anaerobic conditions

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.

microbiology↗