Biology subjects

Rismondo, J.

Publications and source records attributed to Rismondo, J..

17 recordsLinked to original sources

bioRxiv2026

No two are the same: Identification and characterization of novel hyperactive PrfA* variants in Listeria monocytogenes

The virulence potential of the human pathogen Listeria monocytogenes is primarily controlled by the master virulence regulator PrfA. PrfA activity is tightly regulated to prevent virulence gene expression outside the host, while enabling their rapid induction upon host cell entry. One mechanism controlling PrfA activity is the sugar-dependent inhibition. Readily metabolizable sugars, such as glucose or cellobiose, inhibit PrfA; however, the precise mechanism and the factors involved in this regulation remain unknown. Here, a suppressor screen was performed to identify these factors. Although the screen did not reveal any factors mediating the sugar-dependent inhibition of PrfA, it resulted in the isolation of 13 novel hyperactive PrfA variants, termed PrfA*. All newly identified PrfA* variants were characterized using growth, PlcB activity and promoter activity assays. Interestingly, two of these PrfA* variants, PrfAY126F and PrfAV153M, were shown to function even in the absence of PrfAs cofactor glutathione. In addition, a subset of variants was analyzed for intracellular replication using in vitro infection assays. Across all assays, the effect of the PrfA* mutations were found to be highly variant- and gene-dependent, with no two variants exhibiting identical phenotypes. Importantly, these results demonstrate that the transcriptional and phenotypic consequences of PrfA* mutations cannot be reliably predicted from a single assay. Thus, comprehensive characterization using multiple complementary approaches is essential to determine the effects of PrfA* mutations on the expression of PrfA-regulated genes and the pathogenic potential of L. monocytogenes strains carrying these mutations.

microbiology↗

bioRxiv2026

pTRIP, a novel integration plasmid for Listeria monocytogenes

In the past decades, several tools to genetically modify the human pathogen Listeria monocytogenes were developed. Here, we constructed a new integrative plasmid system for L. monocytogenes named pTRIP, for treB insertion plasmid. pTRIP is a vector which stably integrates into the treB locus of the wild type EGD-e. This locus encodes the sole trehalose-specific EIIB and EIIC component of a phosphotransferase system. Successful integration leads to the disruption of treB and thus, to an inability of the resulting L. monocytogenes strains to grow on trehalose as sole carbon source. Due to integration through double homologous recombination, it is the first integrative system which does not require antibiotic selection pressure. To assess functionality of the pTRIP system, prfA and its native promoter region were integrated into the treB locus of a {Delta}prfA strain. Complementation was confirmed in 78% of the isolated clones, indicating successful integration of prfA into the treB locus. We further constructed derivatives of pTRIP harboring the constitutive Pp60 (pTRIP1) and the inducible Prha (pTRIP2) promoter to further expand application possibilities. Microscopic analyses confirmed the functionality of both promoter constructs and showed dose-dependent induction for Prha. pTRIP is an efficient tool for stable gene expression as well as functional studies and expands genetic modification possibilities for L. monocytogenes.

microbiology↗

bioRxiv2026

Trehalose metabolism and its impact on PrfA activity in Listeria monocytogenes

Listeria monocytogenes can grow as a saprophyte on decaying plant material, but can also switch to a pathogenic lifestyle. This switch is mediated by the virulence regulator PrfA, which activates the expression of most virulence genes. PrfA activity is tightly regulated by several mechanisms to ensure that virulence genes are only expressed within the host. One of these regulatory mechanisms is the sugar-dependent repression. In the presence of readily metabolizable sugars, which are imported via phosphotransferase systems (PTS) such as cellobiose, PrfA is repressed; however, the precise mechanism is still unknown. Using a sugar screen, trehalose was identified as the first PTS-dependent sugar that supports growth of L. monocytogenes, but does not seem to impact PrfA activity. We demonstrated that the PTS permease TreB is the sole trehalose importer. After import, trehalose-6-phosphate is cleaved by the phosphotrehalase TreA; however, loss of TreA does not fully abolish growth on trehalose suggesting that L. monocytogenes encodes an additional phosphotrehalase. 13C-Labeling experiments revealed that trehalose metabolism is repressed in the presence of glucose, while it can be metabolized in the presence of glycerol. Additionally, these experiments provided evidence that trehalose and cellobiose are metabolized via identical pathways, including glycolysis and the incomplete TCA cycle, although trehalose has a slower uptake and/or metabolization rate. We therefore hypothesize that sugar-dependent PrfA repression correlates with sugar transport and/or consumption rates, potentially due to varying availability of phosphoenolpyruvate (PEP), which serves as both a metabolic intermediate and phosphate donor for PTS-dependent transport.

microbiology↗

bioRxiv2026

On the role of cell chaining in the attenuation of a Listeria monocytogenes divIVA mutant

Listeria monocytogenes is a facultative intracellular human pathogen capable of invading non-phagocytic host cells, replicating within their cytosol, and spreading directly from cell to cell. These processes are mediated by specialized virulence factors but also depend on the DivIVA protein. DivIVA aids in the secretion of peptidoglycan-degrading autolysins in a process that requires the accessory secretion ATPase SecA2, thereby promoting daughter cell separation following cytokinesis. Consequently, a {Delta}divIVA mutant forms elongated chains of unseparated daughter cells, which may explain its attenuated virulence. To further explore the role of cell chaining for attenuation, we here investigated how different morphological aberrations affect the virulence of L. monocytogenes. To this end, we generated coccoid mreB and filamentous ezrA mutants, and compared them to the {Delta}divIVA mutant in different in vitro infection assays. Coccoid or filamentous morphologies did not impair host cell invasion or intracellular replication, unlike cell chaining of the {Delta}divIVA mutant. Introduction of a hyperactive allele of the PrfA virulence regulator, resulting in constitutive overexpression of virulence genes, was sufficient to restore the invasion defect of the {Delta}divIVA mutant, despite its pronounced cell chaining phenotype, but did not recover intracellular replication. We isolated suppressors of the {Delta}divIVA mutant carrying mutations in secA2, which likely enhance the SecA2 ATPase activity. In these suppressors, autolysin secretion, daughter cell separation, and invasion were fully restored, and intracellular replication was partially recovered. Thus, maintaining normal rod-shaped morphology plays a minor role in L. monocytogenes pathogenesis. Instead, virulence attenuation in the {Delta}divIVA mutant is better explained by distortions in PrfA- and SecA2-dependent processes. Author SummaryListeria monocytogenes infects humans by entering host cells, replicating intracellularly, and spreading from infected cells to neighboring cells. The bacterium is rod-shaped and normally occurs as single or double cells that can efficiently complete all stages of infection. However, a class of virulence-attenuated L. monocytogenes mutants forms long cell chains, suggesting a link between cell morphology and virulence. One such mutant lacks the cell division protein DivIVA and is severely impaired in infection, as it neither invades host cells, replicates in the cytoplasm nor spreads from cell to cell. To determine whether virulence attenuation is caused by cell chaining itself or by additional functional defects, we compared the virulence of the divIVA mutant with that of mutants exhibiting other morphological abnormalities. In addition, we isolated suppressor mutations that restore normal cell separation in the divIVA mutant and examined their effects on virulence. Our results show that cell morphology per se does not generally determine listerial virulence. Instead, they indicate that specific virulence-related functions are impaired in chain-forming L. monocytogenes mutants. These findings advance our understanding of the virulence defects associated with cell chaining and provide a basis for identifying their real underlying molecular causes.

microbiology↗

bioRxiv2025

Cysteine-rich receptor-like kinases mediate Wall Teichoic Acid perception in Arabidopsis

Plants and animals detect microbe-associated molecular patterns (MAMPs) to initiate defense responses. Both lineages employ pattern recognition receptors (PRRs), yet plant mechanisms for sensing Gram-positive bacterial MAMPs remain poorly understood. Wall teichoic acids (WTAs), anionic glycopolymers unique to Gram-positive bacteria, are shown here to activate salicylic acid signaling, defense priming, and a non-canonical programmed cell death in Arabidopsis. We demonstrate that glycosylated WTAs from diverse Gram-positive species elicit these responses and provide genetic evidence that their recognition depends on two members of the plant-specific CYSTEINE-RICH RECEPTOR-LIKE KINASE (CRK) gene family, which is absent in animals and whose function remains elusive. These findings reveal WTAs as a novel class of MAMPs in plants and highlight CRKs as key components in their perception.

plant biology↗

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

Adaptation mechanisms of Listeria monocytogenes to quaternary ammonium compounds

Listeria monocytogenes is ubiquitously found in nature and can easily enter food-processing facilities due to contaminations of raw materials. Several countermeasures are used to combat contamination of food products, for instance the use of disinfectants that contain quaternary ammonium compounds, such as benzalkonium chloride (BAC) and cetyltrimethylammonium bromide (CTAB). In this study, we assessed the potential of the commonly used wildtype strain EGD-e to adapt to BAC and CTAB under laboratory growth conditions. All BAC-tolerant suppressors exclusively carried mutations in fepR or its promoter region likely resulting in the overproduction of the efflux pump FepA. In contrast, CTAB- tolerance was associated with mutations in sugR, which regulates the expression of the efflux pumps SugE1 and SugE2. L. monocytogenes strains lacking either FepA or SugE1/2 could still acquire tolerance towards BAC and CTAB. Genomic analysis revealed that the overproduction of the remaining efflux system could compensate for the deleted one. Even in the absence of both efflux systems, tolerant strains could be isolated, which all carried mutations in the diacylglycerol kinase encoding gene lmo1753 (dgkB). DgkB converts diacylglycerol to phosphatidic acid, which is subsequently re-used for the synthesis of phospholipids suggesting that alterations in membrane composition could be the third adaptation mechanism. Originality-Significance StatementSurvival and proliferation of Listeria monocytogenes in the food industry is an ongoing concern, and while there are various countermeasures to combat contamination of food products, the pathogen still successfully manages to withstand the harsh conditions present in food-processing facilities, resulting in reoccurring outbreaks, subsequent infection, and disease. To counteract the spread of L. monocytogenes it is crucial to understand and elucidate the underlying mechanism that permit their successful evasion. We here present various adaptation mechanisms of L. monocytogenes to withstand two important quaternary ammonium compounds.

microbiology↗

bioRxiv2023

Physiological studies on pH and salt tolerance and the utilization of diverse carbon sources by Listeria monocytogenes

The foodborne pathogen Listeria monocytogenes can grow in a wide range of environmental conditions. For the study of the physiology of this organism, several chemically defined media have been developed over the past decades. Here, we examined the ability of L. monocytogenes wildtype strains EGD-e and 10403S to grow under salt and pH stress in Listeria synthetic medium (LSM). Furthermore, we determined that a wide range of carbon sources could support growth of both wildtype strains in LSM. However, for hexose phosphate sugars such as glucose-1-phosphate, both L. monocytogenes strains need to be pre-grown under conditions, where the major virulence regulator PrfA is active. In addition, growth of both L. monocytogenes strains was observed when LSM was supplemented with the amino acid sugar N-acetylmannosamine (ManNAc). We were able to show that some of the proteins encoded in the operon lmo2795-nanE, such as the ManNAc-6-phosphate epimerase NanE, are required for growth in presence of ManNAc. The first gene of the operon, lmo2795, encodes a transcriptional regulator of the RpiR family. Using electrophoretic mobility shift assays and quantitative real time PCR analysis, we were able to show that Lmo2795 binds to the promoter region of the operon lmo2795-nanE and activates its expression. Originality-Significance StatementCurrent knowledge of growth and survival of the human pathogen Listeria monocytogenes under diverse stress conditions is mostly generated in complex medium, a condition that is rarely found in the environment or host of the pathogen. Our work contributes to the characterization of the physiology of L. monocytogenes grown under nutrient limiting conditions and its growth requirements with regards to metabolizable carbon sources.

microbiology↗

bioRxiv2022

Imbalance of peptidoglycan biosynthesis alters the cell surface charge of Listeria monocytogenes

The bacterial cell wall is composed of a thick layer of peptidoglycan and cell wall polymers, which are either embedded in the membrane or linked to the peptidoglycan backbone and referred to as lipoteichoic acid (LTA) and wall teichoic acid (WTA), respectively. Modifications of the peptidoglycan or WTA backbone can alter the susceptibility of the bacterial cell towards cationic antimicrobials and lysozyme. The human pathogen Listeria monocytogenes is intrinsically resistant towards lysozyme, mainly due to deacetylation and O-acetylation of the peptidoglycan backbone via PgdA and OatA. Recent studies identified additional factors, which contribute to the lysozyme resistance of this pathogen. One of these is the predicted ABC transporter, EslABC. An eslB mutant is hyper-sensitive towards lysozyme, likely due to the production of thinner and less O-acetylated peptidoglycan. Using a suppressor screen, we show here that suppression of eslB phenotypes could be achieved by enhancing peptidoglycan biosynthesis, reducing peptidoglycan hydrolysis or alterations in WTA biosynthesis and modification. The lack of EslB also leads to a higher negative surface charge, which likely stimulates the activity of peptidoglycan hydrolases and lysozyme. Based on our results, we hypothesize that the portion of cell surface exposed WTA is increased in the eslB mutant due to the thinner peptidoglycan layer and that latter one could be caused by an impairment in UDP-N-acetylglucosamine (UDP-GlcNAc) production or distribution.

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

The YtrBCDEF ABC transporter is involved in the control of social activities in Bacillus subtilis

Bacillus subtilis develops genetic competence for the uptake of foreign DNA when cells enter the stationary phase and a high cell density is reached. These signals are integrated by the competence transcription factor ComK which is subject to transcriptional, post-transcriptional and post-translational regulation. Many proteins are involved in the development of competence, both to control ComK activity and to mediate DNA uptake. However, the precise function they play in competence development is often unknown. In this study, we have tested whether proteins required for genetic transformation play a role in the activation of ComK or rather downstream of competence gene expression. While these possibilities could be distinguished for most of the tested factors, two proteins (PNPase and the transcription factor YtrA) are required both for full ComK activity and for the downstream processes of DNA uptake and integration. Further analyses of the role of the transcription factor YtrA for the competence development revealed that the constitutive expression of the YtrBCDEF ABC transporter in the ytrA mutant causes the loss of genetic competence. Moreover, constitutive expression of this ABC transporter also interferes with biofilm formation. Since the ytrGABCDEF operon is induced by cell wall-targeting antibiotics, we tested the cell wall properties upon overexpression of the ABC transporter and observed an increased thickness of the cell wall. The composition and properties of the cell wall are important for competence development and biofilm formation, suggesting that the increased cell wall thickness as a result of YtrBCDEF overexpression causes the observed phenotypes.

microbiology↗

bioRxiv2020

EslB is required for cell wall Integrity and cell division in Listeria monocytogenes

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. IMPORTANCEThe 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 affects lysozyme resistance, but also endogenous cell lysis, cell wall biosynthesis, cell division and the ability of the bacterium to grow in media containing high concentrations of sugars. Our results indicate that EslB is by a yet unknown mechanism an important determinant for cell wall integrity in L. monocytogenes.

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

Bacterial cell wall integrity surveillance is connected to peptidoglycan biosynthesis by the essential phosphorylation of ReoM

Peptidoglycan (PG) is the main component of bacterial cell walls and the target for many antibiotics. PG biosynthesis is tightly coordinated with cell wall growth and turnover, and many of these control activities depend upon PASTA-domain containing eukaryotic-like serine/threonine protein kinases (PASTA-eSTK) that sense PG fragments. However, only a few PG biosynthetic enzymes are direct kinase substrates. Here, we identify the conserved ReoM protein as a novel PASTA-eSTK substrate in the Gram-positive pathogen Listeria monocytogenes. Our data show that the phosphorylation of ReoM is essential as it controls ClpCP-dependent proteolytic degradation of the essential enzyme MurA, which catalyses the first committed step in PG biosynthesis. We also identify ReoY as a second novel factor required for degradation of ClpCP substrates. Collectively, our data imply that the first committed step of PG biosynthesis is activated through control of ClpCP protease activity in response to signals of PG homeostasis imbalance.

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

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

The cell cycle regulator GpsB functions as cytosolic adaptor for multiple cell wall enzymes

Bacterial growth and cell division requires precise spatiotemporal regulation of the synthesis and remodelling of the peptidoglycan layer that surrounds the cytoplasmic membrane. GpsB is a cytosolic protein that affects cell wall synthesis by binding to the cytoplasmic mini-domains of peptidoglycan synthases to ensure their correct subcellular localisation. Here we have discovered critical structural features for the interaction of GpsB with peptidoglycan synthases from three different bacteria and demonstrated their importance for cell wall growth and viability. We have used these structural motifs to predict and confirm novel partners of GpsB in Bacillus subtilis, illuminating the role of this key regulator of peptidoglycan synthesis. GpsB thus functions as an adaptor, to mediate the interaction between membrane proteins, scaffolding proteins, signalling proteins and enzymes to generate larger protein complexes at specific sites in a bacterial cell cycle-dependent manner. Given the importance of GpsB in pathogenic bacteria, this study has not only revealed mechanistic details of how cell wall synthesis is co-ordinated with the bacterial cell cycle but could also represent a starting point for the design of much needed new antibiotics.

microbiology↗