Listeria monocytogenes genes supporting growth under standard laboratory cultivation conditions and during macrophage infection.

The Gram-positive bacterium Listeria monocytogenes occurs widespread in the environment and infects humans when ingested along with contaminated food. Such infections are particularly dangerous for risk group patients, for whom they represent a life-threatening disease. To invent novel strategies to control contamination and disease, it is important to identify those cellular processes that maintain pathogen growth inside and outside the host. Here, we have applied transposon insertion sequencing (Tn-Seq) to L. monocytogenes for the identification of such processes on a genome-wide scale. Our approach identified 394 open reading frames that are required for growth under standard laboratory conditions and 42 further genes, which become necessary during intracellular growth in macrophages. Most of these genes encode components of the translation machinery and act in chromosome-related processes, cell division, and biosynthesis of the cellular envelope. Several cofactor biosynthesis pathways and 29 genes with unknown functions are also required for growth, suggesting novel options for the development of antilisterial drugs. Among the genes specifically required during intracellular growth are known virulence factors, genes compensating intracellular auxotrophies, and several cell division genes. Our experiments also highlight the importance of PASTA kinase signaling for general viability and of glycine metabolism and chromosome segregation for efficient intracellular growth of L. monocytogenes.

Tartrolon sensing and detoxification by the Listeria monocytogenes timABR resistance operon.

Listeria monocytogenes is a foodborne bacterium that naturally occurs in the soil. Originating from there, it contaminates crops and infects farm animals and their consumption by humans may lead to listeriosis, a systemic life-threatening infectious disease. The adaptation of L. monocytogenes to such contrastive habitats is reflected by the presence of virulence genes for host infection and other genes for survival under environmental conditions. Among the latter are ABC transporters for excretion of antibiotics produced by environmental competitors; however, most of these transporters have not been characterized. Here, we generated a collection of promoter-lacZ fusions for genes encoding ABC-type drug transporters of L. monocytogenes and screened this reporter strain collection for induction using a library of natural compounds produced by various environmental microorganisms. We found that the timABR locus (lmo1964-lmo1962) was induced by the macrodiolide antibiotic tartrolon B, which is synthesized by the soil myxobacterium Sorangium cellulosum. Tartrolon B resistance of L. monocytogenes was dependent on timAB, encoding the ATPase and the permease component of a novel ABC transporter. Moreover, transplantation of timAB was sufficient to confer tartrolon B resistance to Bacillus subtilis. Expression of the timABR locus was found to be auto-repressed by the TimR repressor, whose repressing activity was lost in the presence of tartrolon B. We also demonstrate that tartrolon sensitivity was suppressed by high external potassium concentrations, suggesting that tartrolon acts as potassium ionophore. Our results help to map the ecological interactions of an important human pathogen with its co-residing species within their joint natural reservoir.

MurA escape mutations uncouple peptidoglycan biosynthesis from PrkA signaling.

Gram-positive bacteria are protected by a thick mesh of peptidoglycan (PG) completely engulfing their cells. This PG network is the main component of the bacterial cell wall, it provides rigidity and acts as foundation for the attachment of other surface molecules. Biosynthesis of PG consumes a high amount of cellular resources and therefore requires careful adjustments to environmental conditions. An important switch in the control of PG biosynthesis of Listeria monocytogenes, a Gram-positive pathogen with a high infection fatality rate, is the serine/threonine protein kinase PrkA. A key substrate of this kinase is the small cytosolic protein ReoM. We have shown previously that ReoM phosphorylation regulates PG formation through control of MurA stability. MurA catalyzes the first step in PG biosynthesis and the current model suggests that phosphorylated ReoM prevents MurA degradation by the ClpCP protease. In contrast, conditions leading to ReoM dephosphorylation stimulate MurA degradation. How ReoM controls degradation of MurA and potential other substrates is not understood. Also, the individual contribution of the ~20 other known PrkA targets to PG biosynthesis regulation is unknown. We here present murA mutants which escape proteolytic degradation. The release of MurA from ClpCP-dependent proteolysis was able to activate PG biosynthesis and further enhanced the intrinsic cephalosporin resistance of L. monocytogenes. This latter effect required the RodA3/PBP B3 transglycosylase/transpeptidase pair. One murA escape mutation not only fully rescued an otherwise non-viable prkA mutant during growth in batch culture and inside macrophages but also overcompensated cephalosporin hypersensitivity. Our data collectively indicate that the main purpose of PrkA-mediated signaling in L. monocytogenes is control of MurA stability during standard laboratory growth conditions and intracellular growth in macrophages. These findings have important implications for the understanding of PG biosynthesis regulation and ß-lactam resistance of L. monocytogenes and related Gram-positive bacteria.

ListiWiki: A database for the foodborne pathogen Listeria monocytogenes.

Listeria monocytogenes is a Gram positive foodborne pathogen that regularly causes outbreaks of systemic infectious diseases. The bacterium maintains a facultative intracellular lifestyle; it thrives under a variety of environmental conditions and is able to infect human host cells. L. monocytogenes is genetically tractable and therefore has become an attractive model system to study the mechanisms employed by facultative intracellular bacteria to invade eukaryotic cells and to replicate in their cytoplasm. Besides its importance for basic research, L. monocytogenes also serves as a paradigmatic pathogen in genomic epidemiology, where the relative stability of its genome facilitates successful outbreak detection and elucidation of transmission chains in genomic pathogen surveillance systems. In both terms, it is necessary to keep the annotation of the L. monocytogenes genome up to date. Therefore, we have created the database ListiWiki (http://listiwiki.uni-goettingen.de/) which stores comprehensive information on the widely used L. monocytogenes reference strain EDG-e. ListiWiki is designed to collect information on genes, proteins and RNAs and their relevant functional characteristics, but also further information such as mutant phenotypes, available biological material, and publications. In its present form, ListiWiki combines the most recent annotation of the EDG-e genome with published data on gene essentiality, gene expression and subcellular protein localization. ListiWiki also predicts protein-protein interactions networks based on protein homology to Bacillus subtilis proteins, for which detailed interaction maps have been compiled in the sibling database SubtiWiki. Furthermore, crystallographic information of proteins is made accessible through integration of Protein Structure Database codes and AlphaFold structure predictions. ListiWiki is an easy-to-use web interface that has been developed with a focus on an intuitive access to all information. Use of ListiWiki is free of charge and its content can be edited by all members of the scientific community after registration. In our labs, ListiWiki has already become an important and easy to use tool to quickly access genome annotation details that we can keep updated with advancing knowledge. It also might be useful to promote the comprehensive understanding of the physiology and virulence of an important human pathogen.

Adaptation of Listeria monocytogenes to perturbation of c-di-AMP metabolism underpins its role in osmoadaptation and identifies a fosfomycin uptake system.

The human pathogen Listeria monocytogenes synthesizes and degrades c-di-AMP using the diadenylate cyclase CdaA and the phosphodiesterases PdeA and PgpH respectively. c-di-AMP is essential because it prevents the uncontrolled uptake of osmolytes. Here, we studied the phenotypes of cdaA, pdeA, pgpH and pdeA pgpH mutants with defects in c-di-AMP metabolism and characterized suppressor mutants restoring their growth defects. The characterization of the pdeA pgpH mutant revealed that the bacteria show growth defects in defined medium, a phenotype that is invariably suppressed by mutations in cdaA. The previously reported growth defect of the cdaA mutant in rich medium is suppressed by mutations that osmotically stabilize the c-di-AMP-free strain. We also found that the cdaA mutant has an increased sensitivity against isoleucine. The isoleucine-dependent growth inhibition of the cdaA mutant is suppressed by codY mutations that likely reduce the DNA-binding activity of encoded CodY variants. Moreover, the characterization of the cdaA suppressor mutants revealed that the Opp oligopeptide transport system is involved in the uptake of the antibiotic fosfomycin. In conclusion, the suppressor analysis corroborates a key function of c-di-AMP in controlling osmolyte homeostasis in L. monocytogenes.

Elements in the LftR Repressor Operator Interface Contributing to Regulation of Aurantimycin Resistance in Listeria monocytogenes.

The bacterium Listeria monocytogenes ubiquitously occurs in the environment but can cause severe invasive disease in susceptible individuals when ingested. We recently identified the L. monocytogenes genes lieAB and lftRS, encoding a multidrug resistance ABC transporter and a regulatory module, respectively. These genes jointly mediate resistance against aurantimycin, an antibiotic produced by the soil-dwelling species Streptomyces aurantiacus, and thus contribute to the survival of L. monocytogenes in its natural habitat, the soil. Repression of lieAB and lftRS is exceptionally tight but strongly induced in the presence of aurantimycin. Repression depends on LftR, which belongs to subfamily 2 of the PadR-like transcriptional repressors. To better understand this interesting class of transcriptional repressors, we here deduce the LftR operator sequence from a systematic truncation and mutation analysis of the P lieAB promoter. The sequence identified is also present in the P lftRS promoter but not found elsewhere in the chromosome. Mutational analysis of the putative operator in the P lftRS promoter confirmed its relevance for LftR-dependent repression. The proposed operator sequence was sufficient for DNA binding by LftR in vitro, and a mutation in this sequence affected aurantimycin resistance. Our results provide further insights into the transcriptional adaptation of an important human pathogen to survive the conditions in its natural reservoir.IMPORTANCEListeria monocytogenes is an environmental bacterium that lives in the soil but can infect humans upon ingestion, and this can lead to severe invasive disease. Adaptation to these entirely different habitats involves massive reprogramming of transcription. Among the differentially expressed genes is the lieAB operon, which encodes a transporter for the detoxification of aurantimycin, an antimicrobial compound produced by soil-dwelling competitors. While lieAB is important for survival in the environment, its expression is detrimental during infection. We here identify critical elements in the lieAB promoter and its transcriptional regulator LftR that contribute to habitat-specific expression of the lieAB genes. These results further clarify the molecular mechanisms underlying the aurantimycin resistance of L. monocytogenes.

Ongoing High Incidence and Case-Fatality Rates for Invasive Listeriosis, Germany, 2010-2019.

We used 10 years of surveillance data to describe listeriosis frequency in Germany. Altogether, 5,576 cases were reported, 91% not pregnancy associated; case counts increased over time. Case-fatality rate was 13% in non-pregnancy-associated cases, most in adults ≥65 years of age. Detecting, investigating, and ending outbreaks might have the greatest effect on incidence.

Large Nationwide Outbreak of Invasive Listeriosis Associated with Blood Sausage, Germany, 2018-2019.

Invasive listeriosis is a severe foodborne infection in humans and is difficult to control. Listeriosis incidence is increasing worldwide, but some countries have implemented molecular surveillance programs to improve recognition and management of listeriosis outbreaks. In Germany, routine whole-genome sequencing, core genome multilocus sequence typing, and single nucleotide polymorphism calling are used for subtyping of Listeria monocytogenes isolates from listeriosis cases and suspected foods. During 2018-2019, an unusually large cluster of L. monocytogenes isolates was identified, including 134 highly clonal, benzalkonium-resistant sequence type 6 isolates collected from 112 notified listeriosis cases. The outbreak was one of the largest reported in Europe during the past 25 years. Epidemiologic investigations identified blood sausage contaminated with L. monocytogenes highly related to clinical isolates; withdrawal of the product from the market ended the outbreak. We describe how epidemiologic investigations and complementary molecular typing of food isolates helped identify the outbreak vehicle.

Structural basis for interaction of DivIVA/GpsB proteins with their ligands.

DivIVA proteins and their GpsB homologues are late cell division proteins found in Gram-positive bacteria. DivIVA/GpsB proteins associate with the inner leaflet of the cytosolic membrane and act as scaffolds for other proteins required for cell growth and division. DivIVA/GpsB proteins comprise an N-terminal lipid-binding domain for membrane association fused to C-terminal domains supporting oligomerization. Despite sharing the same domain organization, DivIVA and GpsB serve different cellular functions: DivIVA plays diverse roles in division site selection, chromosome segregation and controlling peptidoglycan homeostasis, whereas GpsB contributes to the spatiotemporal control of penicillin-binding protein activity. The crystal structures of the lipid-binding domains of DivIVA from Bacillus subtilis and GpsB from several species share a fold unique to this group of proteins, whereas the C-terminal domains of DivIVA and GpsB are radically different. A number of pivotal features identified from the crystal structures explain the functional differences between the proteins. Herein we discuss these structural and functional relationships and recent advances in our understanding of how DivIVA/GpsB proteins bind and recruit their interaction partners, knowledge that might be useful for future structure-based DivIVA/GpsB inhibitor design.

Aurantimycin resistance genes contribute to survival of Listeria monocytogenes during life in the environment.

Bacteria can cope with toxic compounds such as antibiotics by inducing genes for their detoxification. A common detoxification strategy is compound excretion by ATP-binding cassette (ABC) transporters, which are synthesized upon compound contact. We previously identified the multidrug resistance ABC transporter LieAB in Listeria monocytogenes, a Gram-positive bacterium that occurs ubiquitously in the environment, but also causes severe infections in humans upon ingestion. Expression of the lieAB genes is strongly induced in cells lacking the PadR-type transcriptional repressor LftR, but compounds leading to relief of this repression in wild-type cells were not known. Using RNA-Seq and promoter-lacZ fusions, we demonstrate highly specific repression of the lieAB and lftRS promoters through LftR. Screening of a natural compound library yielded the depsipeptide aurantimycin A - synthesized by the soil-dwelling Streptomyces aurantiacus - as the first known naturally occurring inducer of lieAB expression. Genetic and phenotypic experiments concordantly show that aurantimycin A is a substrate of the LieAB transporter and thus, lftRS and lieAB represent the first known genetic module conferring and regulating aurantimycin A resistance. Collectively, these genes may support the survival of L. monocytogenes when it comes into contact with antibiotic-producing bacteria in the soil.

Stimulation of PgdA-dependent peptidoglycan N-deacetylation by GpsB-PBP A1 in Listeria monocytogenes.

Listeria monocytogenes and other pathogenic bacteria modify their peptidoglycan to protect it against enzymatic attack through the host innate immune system, such as the cell wall hydrolase lysozyme. During our studies on GpsB, a late cell division protein that controls activity of the bi-functional penicillin binding protein PBP A1, we discovered that GpsB influences lysozyme resistance of L. monocytogenes as mutant strains lacking gpsB showed an increased lysozyme resistance. Deletion of pbpA1 corrected this effect, demonstrating that PBP A1 is also involved in this. Susceptibility to lysozyme mainly depends on two peptidoglycan modifying enzymes: The peptidoglycan N-deacetylase PgdA and the peptidoglycan O-acetyltransferase OatA. Genetic and biochemical experiments consistently demonstrated that the increased lysozyme resistance of the ΔgpsB mutant was PgdA-dependent and OatA-independent. Protein-protein interaction studies supported the idea that GpsB, PBP A1 and PgdA form a complex in L. monocytogenes and identified the regions in PBP A1 and PgdA required for complex formation. These results establish a physiological connection between GpsB, PBP A1 and the peptidoglycan modifying enzyme PgdA. To our knowledge, this is the first reported link between a GpsB-like cell division protein and factors important for escape from the host immune system.

Whole-Genome Sequencing of Recent Listeria monocytogenes Isolates from Germany Reveals Population Structure and Disease Clusters.

Listeria monocytogenes causes foodborne outbreaks with high mortality. For improvement of outbreak cluster detection, the German consiliary laboratory for listeriosis implemented whole-genome sequencing (WGS) in 2015. A total of 424 human L. monocytogenes isolates collected in 2007 to 2017 were subjected to WGS and core-genome multilocus sequence typing (cgMLST). cgMLST grouped the isolates into 38 complexes, reflecting 4 known and 34 unknown disease clusters. Most of these complexes were confirmed by single nucleotide polymorphism (SNP) calling, but some were further differentiated. Interestingly, several cgMLST cluster types were further subtyped by pulsed-field gel electrophoresis, partly due to phage insertions in the accessory genome. Our results highlight the usefulness of cgMLST for routine cluster detection but also show that cgMLST complexes require validation by methods providing higher typing resolution. Twelve cgMLST clusters included recent cases, suggesting activity of the source. Therefore, the cgMLST nomenclature data presented here may support future public health actions.

Suppressor Mutations Linking gpsB with the First Committed Step of Peptidoglycan Biosynthesis in Listeria monocytogenes.

The cell division protein GpsB is a regulator of the penicillin binding protein A1 (PBP A1) in the Gram-positive human pathogen Listeria monocytogenes Penicillin binding proteins mediate the last two steps of peptidoglycan biosynthesis as they polymerize and cross-link peptidoglycan strands, the main components of the bacterial cell wall. It is not known what other processes are controlled by GpsB. L. monocytogenes gpsB mutants are unable to grow at 42°C, but we observed that spontaneous suppressors correcting this defect arise on agar plates with high frequency. We here describe a first set of gpsB suppressors that mapped to the clpC and murZ genes. While ClpC is the ATPase component of the Clp protease, MurZ is a paralogue of the listerial UDP-N-acetylglucosamine (UDP-GlcNAc) 1-carboxyvinyltransferase MurA. Both enzymes catalyze the enolpyruvyl transfer from phosphoenolpyruvate to UDP-GlcNAc, representing the first committed step of peptidoglycan biosynthesis. We confirmed that clean deletion of the clpC or murZ gene suppressed the ΔgpsB phenotype. It turned out that the absence of either gene leads to accumulation of MurA, and we show that artificial overexpression of MurA alone was sufficient for suppression. Inactivation of other UDP-GlcNAc-consuming pathways also suppressed the heat-sensitive growth of the ΔgpsB mutant, suggesting that an increased influx of precursor molecules into peptidoglycan biosynthesis can compensate for the lack of GpsB. Our results support a model according to which PBP A1 becomes misregulated and thus toxic in the absence of GpsB due to unproductive consumption of cell wall precursor molecules. IMPORTANCE: The late cell division protein GpsB is important for cell wall biosynthesis in Gram-positive bacteria. GpsB of the human pathogen L. monocytogenes interacts with one of the key enzymes of this pathway, penicillin binding protein A1 (PBP A1), and influences its activity. PBP A1 catalyzes the last two steps of cell wall biosynthesis, but it is unknown how GpsB controls PBP A1. We observed that a L. monocytogenes gpsB mutant forms spontaneous suppressors and have mapped their mutations to genes mediating and influencing the first step of cell wall biosynthesis, likely stimulating the influx of metabolites into this pathway. We assume that GpsB is important to ensure productive incorporation of cell wall precursors into the peptidoglycan sacculus by PBP A1.

Genetic Dissection of DivIVA Functions in Listeria monocytogenes.

DivIVA is a membrane binding protein that clusters at curved membrane regions, such as the cell poles and the membrane invaginations occurring during cell division. DivIVA proteins recruit many other proteins to these subcellular sites through direct protein-protein interactions. DivIVA-dependent functions are typically associated with cell growth and division, even though species-specific differences in the spectrum of DivIVA functions and their causative interaction partners exist. DivIVA from the Gram-positive human pathogen Listeria monocytogenes has at least three different functions. In this bacterium, DivIVA is required for precise positioning of the septum at midcell, it contributes to the secretion of autolysins required for the breakdown of peptidoglycan at the septum after the completion of cell division, and it is essential for flagellar motility. While the DivIVA interaction partners for control of division site selection are well established, the proteins connecting DivIVA with autolysin secretion or swarming motility are completely unknown. We set out to identify divIVA alleles in which these three DivIVA functions could be separated, since the question of the degree to which the three functions of L. monocytogenes DivIVA are interlinked could not be answered before. Here, we identify such alleles, and our results show that division site selection, autolysin secretion, and swarming represent three discrete pathways that are independently influenced by DivIVA. These findings provide the required basis for the identification of DivIVA interaction partners controlling autolysin secretion and swarming in the future.IMPORTANCE DivIVA of the pathogenic bacterium Listeria monocytogenes is a central scaffold protein that influences at least three different cellular processes, namely, cell division, protein secretion, and bacterial motility. How DivIVA coordinates these rather unrelated processes is not known. We here identify variants of L. monocytogenes DivIVA, in which these functions are separated from each other. These results have important implications for the models explaining how DivIVA interacts with other proteins.

Molecular Tracing to Find Source of Protracted Invasive Listeriosis Outbreak, Southern Germany, 2012-2016.

We investigated 543 Listeria monocytogenes isolates from food having a temporal and spatial distribution compatible with that of the invasive listeriosis outbreak occurring 2012-2016 in southern Germany. Using forensic microbiology, we identified several products from 1 manufacturer contaminated with the outbreak genotype. Continuous molecular surveillance of food isolates could prevent such outbreaks.

Structure of the bacterial cell division determinant GpsB and its interaction with penicillin-binding proteins.

Each bacterium has to co-ordinate its growth with division to ensure genetic stability of the population. Consequently, cell division and growth are tightly regulated phenomena, albeit different bacteria utilise one of several alternative regulatory mechanisms to maintain control. Here we consider GpsB, which is linked to cell growth and division in Gram-positive bacteria. ΔgpsB mutants of the human pathogen Listeria monocytogenes show severe lysis, division and growth defects due to distortions of cell wall biosynthesis. Consistent with this premise, GpsB interacts both in vitro and in vivo with the major bi-functional penicillin-binding protein. We solved the crystal structure of GpsB and the interaction interfaces in both proteins are identified and validated. The inactivation of gpsB results in strongly attenuated virulence in animal experiments, comparable in degree to classical listerial virulence factor mutants. Therefore, GpsB is essential for in vitro and in vivo growth of a highly virulent food-borne pathogen, suggesting that GpsB could be a target for the future design of novel antibacterials.

Phenotypes Associated with the Essential Diadenylate Cyclase CdaA and Its Potential Regulator CdaR in the Human Pathogen Listeria monocytogenes.

UNLABELLED: Cyclic diadenylate monophosphate (c-di-AMP) is a second messenger utilized by diverse bacteria. In many species, including the Gram-positive human pathogen Listeria monocytogenes, c-di-AMP is essential for growth. Here we show that the single diadenylate cyclase of L. monocytogenes, CdaA, is an integral membrane protein that interacts with its potential regulatory protein, CdaR, via the transmembrane protein domain. The presence of the CdaR protein is not required for the membrane localization and abundance of CdaA. We have also found that CdaR negatively influences CdaA activity in L. monocytogenes and that the role of CdaR is most evident at a high growth temperature. Interestingly, a cdaR mutant strain is less susceptible to lysozyme. Moreover, CdaA contributes to cell division, and cells depleted of CdaA are prone to lysis. The observation that the growth defect of a CdaA depletion strain can be partially restored by increasing the osmolarity of the growth medium suggests that c-di-AMP is important for maintaining the integrity of the protective cell envelope. Overall, this work provides new insights into the relationship between CdaA and CdaR. IMPORTANCE: Cyclic diadenylate monophosphate (c-di-AMP) is a recently identified second messenger that is utilized by the Gram-positive human pathogen Listeria monocytogenes. Here we show that the single diadenylate cyclase of L. monocytogenes, CdaA, is an integral membrane protein that interacts with CdaR, its potential regulatory protein. We show that CdaR is not required for membrane localization or abundance of the diadenylate cyclase, but modulates its activity. Moreover, CdaA seems to contribute to cell division. Overall, this work provides new insights into the relationship between CdaA and CdaR and their involvement in cell growth.

Discrete and overlapping functions of peptidoglycan synthases in growth, cell division and virulence of Listeria monocytogenes.

Upon ingestion of contaminated food, Listeria monocytogenes can cause serious infections in humans that are normally treated with ß-lactam antibiotics. These target Listeria's five high molecular weight penicillin-binding proteins (HMW PBPs), which are required for peptidoglycan biosynthesis. The two bi-functional class A HMW PBPs PBP A1 and PBP A2 have transglycosylase and transpeptidase domains catalyzing glycan chain polymerization and peptide cross-linking, respectively, whereas the three class B HMW PBPs B1, B2 and B3 are monofunctional transpeptidases. The precise roles of these PBPs in the cell cycle are unknown. Here we show that green fluorescent protein (GFP)-PBP fusions localized either at the septum, the lateral wall or both, suggesting distinct and overlapping functions. Genetic data confirmed this view: PBP A1 and PBP A2 could not be inactivated simultaneously, and a conditional double mutant strain is largely inducer dependent. PBP B1 is required for rod-shape and PBP B2 for cross-wall biosynthesis and viability, whereas PBP B3 is dispensable for growth and cell division. PBP B1 depletion dramatically increased ß-lactam susceptibilities and stimulated spontaneous autolysis but had no effect on peptidoglycan cross-linkage. Our in vitro virulence assays indicated that the complete set of all HMW PBPs is required for maximal virulence.

A systematic proteomic analysis of Listeria monocytogenes house-keeping protein secretion systems.

Listeria monocytogenes is a firmicute bacterium causing serious infections in humans upon consumption of contaminated food. Most of its virulence factors are secretory proteins either released to the medium or attached to the bacterial surface. L. monocytogenes encodes at least six different protein secretion pathways. Although great efforts have been made in the past to predict secretory proteins and their secretion routes using bioinformatics, experimental evidence is lacking for most secretion systems. Therefore, we constructed mutants in the main housekeeping protein secretion systems, which are the Sec-dependent transport, the YidC membrane insertases SpoIIIJ and YqjG, as well as the twin-arginine pathway, and analyzed their secretion and virulence defects. Our results demonstrate that Sec-dependent secretion and membrane insertion of proteins via YidC proteins are essential for viability of L. monocytogenes. Depletion of SecA or YidC activity severely affected protein secretion, whereas loss of the Tat-pathway was without any effect on secretion, viability, and virulence. Two-dimensional gel electrophoresis combined with protein identification by mass spectrometry revealed that secretion of many virulence factors and of enzymes synthesizing and degrading the cell wall depends on the SecA route. This finding was confirmed by SecA inhibition experiments using sodium azide. Analysis of secretion of substrates typically dependent on the accessory SecA2 ATPase in wild type and azide resistant mutants of L. monocytogenes revealed for the first time that SecA2-dependent protein secretion also requires the ATPase activity of the house-keeping SecA protein.