Biology subjects

Boeren, S.

Publications and source records attributed to Boeren, S..

18 recordsLinked to original sources

bioRxiv2026

Structural and functional insights into the role of Cysteine-Rich Receptor-Like Kinase 18 (CRK18) in Arabidopsis

Plants perceive and integrate diverse environmental signals through receptor kinase (RK) networks at the plasma membrane. Within this, Cysteine-Rich Receptor-Like Kinases (CRKs) constitute a large but not well-understood subfamily characterised by extracellular domains (ECDs) enriched in cysteine residues. CRKs have been implicated in plant responses to biotic and abiotic stress, as well as in developmental processes. Additionally, several CRKs have been proposed to act as redox sensors. Here, we investigate the homodimerization mechanism of Arabidopsis CRK18 and its regulation by redox conditions. By modulating pH and redox state, we assessed the stability and binding dynamics of the CRK18 ECD and its cysteine mutants. We also tested the plasma membrane localisation of all the cysteine mutants involved in the predicted disulfide bonds, and only CRK18C227A, C228A-ECD resembled the plasma membrane localization of wild-type CRK18. We combine co-immunoprecipitation, Forster resonance energy transfer-fluorescence lifetime imaging microscopy and microscale thermophoresis to quantify CRK18 self-association in planta and in vitro. Furthermore, we place CRK18 dimerization in a broader signalling context by identifying CRK18 interaction partners and CRK18-dependent signalling outputs. Using Arabidopsis CRK18 overexpression lines, we perform (phospho)proteomic and immunoprecipitation-mass spectrometry (IP-MS) analyses to map CRK18-centred signalling networks associated with stress-related responses. The constitutive activation of the CRK18 kinase domain and its interaction with many putative (cell wall) glycan-sensing RKs, including PERK15, suggest a regulatory role for CRK18. The absence of significant changes in the proteome and phosphoproteome of CRK18 overexpression lines in the absence of any trigger, and its restricted mobility after elicitation, suggest that CRK18 requires a stimulus for activation and possibly induces membrane microdomain reorganisation. This is in line with the infection assay with the nematode Heterodera schachtii, which causes modification and targeted damage to plant cell walls during infection, revealing that CRK18 acts as negative regulator of this process.

biochemistry↗

bioRxiv2026

TurboID-based proximity-dependent labeling using SOBIR1 as a bait in potato leads to the identification of novel defense-related signaling partners

The plant immune system comprises a complex signaling network that is activated upon perceiving molecules derived from invading pathogens. The first line of defense at the plant cell surface is mediated by receptor-like proteins (RLPs) and receptor-like kinases (RLKs). RLPs, which lack a cytoplasmic signalling domain themselves, constitutively interact with the RLK SUPRESSOR OF BIR1-1 (SOBIR1), which is a key component initiating immune signal transduction upon pathogen perception. Therefore, elucidating the composition of the SOBIR1 protein complex will contribute to understanding the basic molecular mechanisms of plant disease resistance. Most of the studies focused on the identification of SOBIR1-interacting proteins are limited to model plants, due to technical challenges and lack of reliable genome and proteome databases in crop plants. Here, we evaluate the application of the biotin ligase TurboID (TbID)-based proximity-dependent labeling (PL) approach by transiently expressing SOBIR1 from Nicotiana benthamiana (NbSOBIR1), fused to TbID in leaves of the wild potato Solanum microdontum. We show that NbSOBIR1-YFP-TbID properly accumulates in potato and that proximal proteins are biotinylated. Quantitative proteomic analysis yielded over 130 candidate proteins to be in the proximity of the cytoplasmic kinase domain of NbSOBIR1, of which some could be linked to disease resistance by KEGG pathway and gene ontology (GO) molecular function analysis. We also studied the dynamics of the proteome in proximity of NbSOBIR1 upon perception of the INF1 elicitin of Phytophthora infestans that was co-expressed in potato with the elicitin receptor ELR, which is an RLP that constitutively interacts with SOBIR1. We found more than 80 proteins, including the NB-LRR REQUIRED FOR HR-ASSOCIATED CELL DEATH 1 (NRC1), putatively interacting with NbSOBIR1. In conclusion, we were able to successfully apply PL in potato and a future roadmap for further research on deciphering the composition of protein complexes involved in immune signaling has been established.

plant biology↗

bioRxiv2026

Stressostat cultivation of Lactococcus lactis improves lactate stress resistance through mutations in RNA polymerase

Adaptive laboratory evolution is used to improve the phenotypes of microorganisms and to characterise the mechanisms underlying resistance against complex growth inhibition. Here we focused on lactic acid bacteria (LAB) as starter cultures for food fermentations. Production of LAB starter cultures is challenging due to growth inhibition by organic acids, mainly lactate, produced during fermentation. By utilising stressostat cultivation we generated Lactococcus lactis isolates with enhanced lactate resistance. Using a combination of (meta)genomics, proteomics and pH-controlled batch fermentations, we deciphered the lactate resistance mechanisms of these L. lactis isolates. Proteome responses of L. lactis, combined with similar growth inhibition at high salt, suggest that high lactate mainly causes osmotic stress. We identified RNA polymerase (RNAP) mutations in subunits {beta} (rpoB) and {beta} (rpoC) as key mutations, causing pleiotropic effects in the proteome. These proteome adaptations are linked to enhanced lactate resistance, particularly the resistance to hyperosmotic stress without glycine-betaine supplementation, likely by altering cross-linking of the peptidoglycan in the cell envelope via downregulation of MurE. The proteome changes indicate that lactate might (indirectly) cause oxidative stress. Combined, our study shows that RNAP mutations enhanced lactate resistance through pleotropic effects in the proteome that changed L. lactis responses against multiple stresses. HighlightsO_LILactate resistant variants were isolated from stressostat cultivations C_LIO_LIMetagenomics revealed evolutionary trajectory during stressostat cultivation C_LIO_LIRNAP mutations were identified as key drivers for improved lactate resistance C_LIO_LIRNAP mutations altered the proteome, enhancing the osmotic stress resistance C_LI

microbiology↗

bioRxiv2026

Redox-dependent extracellular interaction networks of Cysteine-Rich Receptor-Like Kinases

Reactive oxygen species (ROS) regulate plant development and immunity, but how extracellular ROS signals are decoded and whether the Cysteine-rich Receptor-like Kinases (CRKs) truly serve as the long-suspected ROS sensors remains uncertain. Here, we combine high-throughput interactomics, redox proteomics, structural modelling, and genetics to map a ROS-dependent CRK interaction landscape in Arabidopsis thaliana. Using a redox-dependent interactome assay (RIACRK) on 40 CRK extracellular domains (ECDs), we identified ROS-modulated dimerisation networks with enhanced inter-community connectivity and hub redistribution in the presence of ROS. Integrating this with developmental and flg22-induced expression profiles reveals spatiotemporally limited subnetworks that likely function during activated immunity and leaf senescence, both of which are associated with extensive ROS production. Differential cysteine alkylation coupled with mass spectrometry shows that cysteines in a subset of CRK ectodomains undergo ROS-dependent oxidation. Notably, solvent-exposed, vicinal cysteines C228/C229 in CRK28 emerge as prime redox-sensitive candidates. CRK28 homodimerises and heterodimerises with CRK17 in vivo, and mutation of C228/C229 retains plasma membrane localisation but abolishes CRK28 homodimerisation, indicating a redox-controlled dimerisation switch. Loss of CRK28 delays senescence, while CRK28 overaccumulation from its native promoter causes dwarfism, premature senescence, autoimmune-like phenotypes, extensive phosphoproteome rewiring, and associations with Pathogenesis Related (PR) proteins, ROS-detoxifying enzymes, receptor(-like) kinases, and vesicle trafficking components. These results indicate that CRK28 is a potential ROS-regulated hub connecting extracellular redox signals to CRK network organisation, immune response, and age-related senescence.

plant biology↗

bioRxiv2026

Effector-centred proximity-dependent labelling enables the discovery of cell-surface immune receptors in plants

Identifying plant disease resistance proteins remains challenging, particularly for cell-surface receptors that perceive apoplastic pathogen effectors through transient or indirect interactions. Here, we establish effector-centred proximity-dependent labelling using TurboID as a protein-level approach to identify immune receptors and guarded host targets in planta. We fused three apoplastic effectors, Avr2 and Avr4 from Fulvia fulva and XEG1 from Phytophthora sojae, to TurboID and transiently expressed these fusion proteins in leaves of Nicotiana benthamiana and tomato (Solanum lycopersicum). The fusions significantly biotinylate their matching receptors, including Cf-4 by Avr4-TurboID and Cf-2 by Avr2-TurboID, as well as the guarded virulence target of Avr2, the Rcr3 protease, demonstrating that both direct and indirect recognition systems can be captured. Application of this approach to XEG1 revealed SlEix1 as the functional ortholog of NbRXEG1. Functional assays and structural modelling support SlEix1 as a signalling-competent receptor contributing to XEG1-triggered immunity. Together with previous studies, these findings position the tomato EIX locus as a multi-effector immune hub encoding closely related receptor-like proteins with distinct ligand specificities. Collectively, this study establishes effector-TurboID-mediated proximity-dependent labelling as a versatile approach for identifying cell surface plant immune receptors and apoplastic virulence targets, providing a scalable route to accelerate resistance gene discovery in crop species.

plant biology↗

bioRxiv2022

An ultra-fast, proteome-wide response to the plant hormone auxin

The plant signaling molecule auxin controls growth and development through a simple nuclear pathway that regulates gene expresion. There are however several cellular and physiological responses to auxin that occur within seconds, far too rapid to be mediated by transcriptional changes, for which no molecular mechanism has yet been identified. Using a phosphoproteomic strategy in Arabidopsis thaliana roots, we identify an ultra-rapid auxin response system that targets over 1700 proteins, many within 30 seconds. Auxin response is chemically specific, requires known auxin-binding proteins, and targets various pathways. Through exploring its temporal dynamics, we infer auxin-triggered kinase-substrate networks and identify apoplastic pH changes as a target of signaling and as part of a relay mechanism. By generating a variety of phosphoproteomic datasets, integrated with structural information in a web-app, and by demonstrating analysis and inference strategies, we offer a resource to explore rapid and dynamic signaling in plants.

plant biology↗

bioRxiv2022

Assessment of infant outgrowth of cow's milk allergy in relation to the faecal microbiome and metaproteome

Previous studies provide evidence for an association between modifications of the gut microbiota in early life and the development of food allergies. We studied the faecal microbiota composition (16S rRNA gene amplicon sequencing) and faecal microbiome functionality (metaproteomics) in a cohort of 40 infants diagnosed with cows milk allergy (CMA) when entering the study. Some of the infants showed outgrowth of CMA after 12 months, while others did not. Faecal microbiota composition of infants was analysed directly after CMA diagnosis (baseline) as well as 6 and 12 months after entering the study. The aim was to gain insight on gut microbiome parameters in relation to outgrowth of CMA. The results of this study show that microbiome differences related to outgrowth of CMA can be mainly identified at the taxonomic level of the 16S rRNA gene, and to a lesser extent at the protein-based microbial taxonomy and functional protein level. At the 16S rRNA gene level outgrowth of CMA is characterized by lower relative abundance of Lachnospiraceae at baseline and lower Bacteroidaceae at visit 12 months.

microbiology↗

bioRxiv2022

Raw milk kefir: microbiota, bioactive peptides, and immune modulation.

This study aims to characterize the microbial and peptidomic composition of raw milk kefir, and to address the potential anti-allergic effects of raw milk kefir using validated research models for food allergy. Raw milk kefir was produced after incubation with a defined freeze-dried starter culture. Kefir was sampled during fermentation at seven-time intervals. For comparison, kefir was also prepared from boiled milk (100 {degrees}C; 5 sec). Peptide compositions were determined for the raw and heated milk, plus kefir end products made from these milks. In a murine food allergy model, the two kefir end products were investigated on their allergy modulating effects. In both kefirs, we identified amplicon sequence variants identical to those in the starter, matching the bacteria Lactococcus lactis, Streptococcus thermophilus, Leuconostoc and the yeast Debaromyces. In raw milk kefir, additional sequence variants of Lactococcus lactis and the yeasts Pichia and Galactomyces could be identified, which were absent in heated milk kefir. Analysis of peptide compositions in kefirs indicated that the number and intensity of peptides drastically increased after fermentation. Heating of milk negatively affected the diversity of the peptide composition in kefir. Only raw milk kefir suppressed the acute allergic skin response to the food allergen ovalbumin in sensitised mice. These effects coincided with differences in the T-cell compartment, with lower percentages of activated Th1 cells and IFNg production after treatment with kefir made from heated milk. The results of this study indicate specific properties of raw milk kefir that may contribute to its additional health benefits.

microbiology↗

bioRxiv2022

Disentangling the lipid divide: Identification of key enzymes for the biosynthesis of unusual Membrane-spanning and Ether lipids in Bacteria

Bacterial membranes are composed of fatty acids (FAs) ester-linked to glycerol-3-phosphate, while archaea possess membranes made of isoprenoid chains ether-linked to glycerol-1-phosphate. Many archaeal species organize their membrane as a monolayer of membrane-spanning lipids (MSLs). Exceptions to this lipid divide are the production by some bacterial species of (ether-bound) MSLs, formed by tail-tail condensation of fatty acids resulting in the formation of (iso) diabolic acids (DAs), which are the likely precursors of paleoclimatological relevant branched glycerol dialkyl glycerol tetraether molecules. However, the enzymes responsible for their production are unknown. Here, we report the discovery of bacterial enzymes responsible for the condensation reaction of fatty acids and for ether bond formation, and confirm that the building blocks of iso-DA are branched iso-FAs. Phylogenomic analyses of the key biosynthetic genes reveal a much wider diversity of potential MSL (ether)-producing bacteria than previously thought, with significant implications for our understanding of the evolution of lipid membranes.

microbiology↗

bioRxiv2021

Manganese modulates metabolic activity and redox homeostasis in translationally-blocked Lactococcus cremoris, impacting metabolic persistence, cell-culturability, and flavor formation.

Manganese (Mn) is an essential trace element that is supplemented in microbial media with varying benefits across species and growth conditions. We found that growth of Lactococcus cremoris was unaffected by manganese omission from the growth medium. The main proteome adaptation to manganese omission involved increased manganese transporter production (up to 2000-fold), while the remaining 10 significant proteome changes were between 1.4 and 4 fold. Further investigation in translationally-blocked (TB), non-growing cells showed that Mn supplementation (20 M) led to approximately 1.5X faster acidification compared to Mn-free conditions. However, this faster acidification stagnated within 24 hours, likely due to draining of intracellular NADH that coincides with substantial loss of culturability. Conversely, without manganese, non-growing cells persisted to acidify for weeks, albeit at a reduced rate, but maintaining redox balance and culturability. Strikingly, despite being unculturable, -keto acid-derived aldehydes continued to accumulate in cells incubated in the presence of manganese, whereas without manganese cells predominantly formed the corresponding alcohols. This is most likely reflecting NADH availability for the alcohol dehydrogenase-catalyzed conversion. Overall, manganese influences the lactococcal acidification rate, and flavor formation capacity in a redox dependent manner. These are important industrial traits especially during cheese ripening, where cells are in a non-growing, often unculturable state.

microbiology↗

bioRxiv2021

Physiological roles of short-chain and long-chain menaquinones (vitamin K2) in Lactococcus cremoris

Lactococcus cremoris and L. lactis are well-known for their occurrence and applications in dairy fermentations, but their niche extends to a range of natural and food production environments. L. cremoris and L. lactis produce MKs (vitamin K2), mainly as the long-chain forms represented by MK-9 and MK-8, and a detectable amount of short-chain forms represented by MK-3. The physiological significance of the different MK forms in the lifestyle of these bacterial species has not been investigated extensively. In this study, we used L. cremoris MG1363 to construct mutants producing different MK profiles by deletion of genes encoding (i) a menaquinone-specific isochorismate synthase, (ii) a geranyltranstransferase and (iii) a prenyl diphosphate synthase. These gene deletions resulted in (i) a non-MK producer ({Delta}menF), (ii) a presumed MK-1 producer ({Delta}ispA) and (iii) a MK-3 producer ({Delta}llmg_0196), respectively. By examining the phenotypes of the MG1363 wildtype strain and respective mutants, including biomass accumulation, stationary phase survival, oxygen consumption, primary metabolites, azo dye/copper reduction, and proteomes, under aerobic, anaerobic and respiration-permissive conditions, we could infer that short-chain MKs like MK-1 and MK-3 are preferred to mediate extracellular electron transfer and reaction with extracellular oxygen, while the long-chain MKs like MK-9 and MK-8 are more efficient in aerobic respiratory electron transport chain. The different electron transfer routes mediated by short-chain and long-chain MKs likely support growth and survival of L. cremoris in a range of (transiently) anaerobic and aerobic niches including food fermentations, highlighting the physiological significance of diverse MKs in L. cremoris.

microbiology↗

bioRxiv2021

Impact of bacterial microcompartment-dependent ethanolamine and propanediol metabolism on Listeria monocytogenes interactions with Caco-2 cells

Bacterial microcompartment (BMC) dependent ethanolamine (eut) and propanediol utilization (pdu) has recently been shown to stimulate anaerobic growth of Listeria monocytogenes. This metabolic repertoire conceivably contributes to the competitive fitness of L. monocytogenes in the human gastrointestinal (GI) tract, where these compounds become available following phospholipid degradation and mucus-derived rhamnose metabolism by commensal microbiota. Previous transcriptomics and mutant studies of eut and pdu L. monocytogenes suggested a possible role of eut and pdu BMC metabolism in transmission in foods and pathogenicity, but data on a potential role of L. monocytogenes interaction with human cells is currently absent. First, we ask which cellular systems are expressed in the activation of eut and pdu BMC metabolism and the extent to which these systems are conserved between the states. We find common and unique systems related to metabolic shifts, stress and virulence factors. Next, we hypothesize that these common and unique activated cellular systems contribute to a role in the interaction of L. monocytogenes interaction with human cells. We present evidence that metabolically primed L. monocytogenes with active eut and pdu BMCs, as confirmed by metabolic analysis, transmission electron microscopy and proteomics, show significantly enhanced translocation efficacy compared to non-induced cells in a trans-well assay using Caco-2 cells, while adhesion and invasion capacity was similar. Taken together, our results provide insights into the possible key cellular players that drive translocation efficacy upon eut and pdu BMC activation.

microbiology↗

bioRxiv2021

Impact of vitamin B12 on rhamnose metabolism, stress defense and in-vitro virulence of Listeria monocytogenes

Listeria monocytogenes is a facultative anaerobe which can cause a severe food-borne infection known as listeriosis. Rhamnose is a deoxyhexose sugar abundant in a range of environments, including the human intestine, and can be degraded by L. monocytogenes in aerobic and anaerobic conditions into lactate, acetate and 1,2-propanediol. Our previous study showed that addition of vitamin B12 stimulates anaerobic growth of L. monocytogenes on rhamnose due to the activation of bacterial microcompartment (BMC)-dependent 1,2-propanediol utilization with concomitant production of propionate and propanol. Notably, anaerobic propanediol metabolism has been linked to virulence of enteric pathogens including Salmonella spp. and L. monocytogenes. In this study we investigate the impact of B12 on aerobic and anerobic growth of L. monocytogenes on rhamnose, and observed growth stimulation and pdu BMC activation only in anaerobically grown cells with B12 added to the medium. Comparative Caco-2 virulence assays, showed that these pdu BMC induced cells have significantly higher translocation efficiency compared to aerobically grown cells (without and with added B12) and non-induced anaerobically grown cells, while adhesion and invasion capacity is similar for all cells. Comparative proteomics analysis showed specific and overlapping responses linked to metabolic shifts, activation of stress defense proteins and virulence factors, with RNA polymerase sigma factor SigL; teichoic acids export ATP-binding protein, TagH; DNA repair and protection proteins RadA and DPS; and glutathione synthase GshAB previously linked to activation of virulence response in L. monocytogenes, uniquely upregulated in anaerobically rhamnose grown pdu BMC induced cells. Our results shed new light into B12 impact on L. monocytogenes competitive fitness and virulence.

microbiology↗

bioRxiv2021

Glycine-Rich RNA-Binding Protein 7 interacts with and potentiates effector-induced immunity by Gpa2 and Rx1 based on an intact RNA Recognition Motif

O_LIThe activity of intracellular plant Nucleotide-Binding Leucine-Rich Repeat (NB-LRR) immune receptors is fine-tuned by interactions between the receptors and their partners. Identifying NB-LRR interacting proteins is, therefore, crucial to advance our understanding of how these receptors function. C_LIO_LIA Co-Immunoprecipitation/Mass-Spectrometry screening was performed in Nicotiana benthamiana to identify host proteins associated with the Gpa2 CC-NB-LRR, which confers resistance against the potato cyst nematode Globodera pallida. A combination of biochemical, cellular, and functional assays was used to assess the role of a candidate interactor in defence. C_LIO_LIA N. benthamiana homolog of the Glycine-Rich RNA-Binding Protein 7 (NbGRP7) protein was prioritized as a novel Gpa2-interacting protein for further investigations. NbGRP7 also associates in planta with the homologous Rx1 receptor, which confers immunity to Potato Virus X. We show that NbGRP7 positively regulates extreme resistance by Rx1 and cell death by Gpa2. Mutating the NbGRP7 RNA recognition motif compromises its role in Rx1-mediated defence. Strikingly, ectopic NbGRP7 expression impacts the steady-state levels of Rx1, which relies on an intact RNA recognition motif. C_LIO_LICombined, our findings illustrate that NbGRP7 is a novel pro-immune component in effector-triggered immunity by regulating Gpa2/Rx1 functioning at a post-transcriptional level. C_LI

plant biology↗

bioRxiv2021

Anaerobic growth of Listeria monocytogenes on rhamnose is stimulated by Vitamin B12 and bacterial microcompartment dependent 1,2-propanediol utilization

The food-borne pathogen Listeria monocytogenes is able to form proteinaceous organelles called bacterial microcompartments (BMCs) that optimize the utilization of substrates, such as 1,2-propanediol, and confer an anaerobic growth advantage. Rhamnose is a deoxyhexose sugar abundant in a range of environments including the human intestine, and can be degraded in anaerobic conditions into 1,2-propanediol, next to acetate and lactate. Rhamnose-derived 1,2-propanediol has been found to link with BMCs in a limited number of commensal human colonic species and some human pathogens such as Salmonella enterica, but the involvement of BMCs in rhamnose metabolism and potential physiological effects on L. monocytogenes are still unknown. In this study, we firstly test the effect of rhamnose uptake and utilization on anaerobic growth of L. monocytogenes EGDe without and with added vitamin B12, followed by metabolic analysis. We unveil that the vitamin B12-dependent activation of pdu stimulates metabolism and anaerobic growth of L. monocytogenes EGDe on rhamnose via 1,2-propanediol degradation into 1-propanol and propionate. Transmission electron microscopy of pdu-induced cells shows that BMCs are formed and additional proteomics experiments confirm expression of pdu BMC shell proteins and enzymes. Finally, we discuss physiological effects and energy efficiency of L. monocytogenes pdu BMC-driven anaerobic rhamnose metabolism and impact on competitive fitness in environments such as the human intestine.

microbiology↗

bioRxiv2020

Bacterial microcompartments linked to the flavin-based extracellular electron transfer drives anaerobic ethanolamine utilization in Listeria monocytogenes

Ethanolamine (EA) is a valuable microbial carbon and nitrogen source derived from phospholipids present in cell membranes. EA catabolism is suggested to occur in so-called bacterial microcompartments (BMCs) and activation of EA utilization (eut) genes is linked to bacterial pathogenesis. Despite reports showing that activation of eut in Listeria monocytogenes is regulated by a vitamin B12-binding riboswitch and that upregulation of eut genes occurs in mice, it remains unknown whether EA catabolism is BMC dependent. Here, we provide evidence for BMC-dependent anaerobic EA utilization via metabolic analysis, proteomics and electron microscopy. First, we show B12-induced activation of the eut operon in L. monocytogenes coupled to uptake and utilization of EA thereby enabling growth. Next, we demonstrate BMC formation in conjunction to EA catabolism with the production of acetate and ethanol in a molar ratio of 2:1. Flux via the ATP generating acetate branch causes an apparent redox imbalance due to reduced regeneration of NAD+ in the ethanol branch resulting in a surplus of NADH. We hypothesize that the redox imbalance is compensated by linking eut BMC to anaerobic flavin-based extracellular electron transfer (EET). Using L. monocytogenes wild type, a BMC mutant and a EET mutant, we demonstrate an interaction between BMC and EET and provide evidence for a role of Fe3+ as an electron acceptor. Taken together, our results suggest an important role of anaerobic BMC-dependent EA catabolism in the physiology of L. monocytogenes, with a crucial role for the flavin-based EET system in redox balancing. IMPORTANCEListeria monocytogenes is a food-borne pathogen causing severe illness and, as such, it is crucial to understand the molecular mechanisms contributing to pathogenicity. One carbon source that allows L. monocytogenes to grow in humans is ethanolamine (EA), which is derived from phospholipids present in eukaryotic cell membranes. It is hypothesized that EA utilization occurs in bacterial microcompartments (BMCs), self-assembling subcellular proteinaceous structures and analogs of eukaryotic organelles. Here, we demonstrate that BMC-driven utilization of EA in L. monocytogenes results in increased energy production essential for anaerobic growth. However, exploiting BMCs and the encapsulated metabolic pathways also requires balancing of oxidative and reductive pathways. We now provide evidence that L. monocytogenes copes with this by linking BMC activity to flavin-based extracellular electron transfer (EET) using iron as an electron acceptor. Our results shed new light on an important molecular mechanism that enables L. monocytogenes to grow using host-derived phospholipid degradation products.

microbiology↗

bioRxiv2020

Proteome constraints reveal targets for improving microbial fitness in nutrient-rich environments

Cells adapt to different conditions via gene expression that tunes metabolism and stress resistance for maximal fitness. Constraints on cellular proteome may limit such expression strategies and introduce trade-offs1; Resource allocation under proteome constraints has emerged as a powerful paradigm to explain regulatory strategies in bacteria2. It is unclear, however, to what extent these constraints can predict evolutionary changes, especially for microorganisms that evolved under nutrient-rich conditions, i.e., multiple available nitrogen sources, such as the lactic acid bacterium Lactococcus lactis. Here we present an approach to identify preferred nutrients from integration of experimental data with a proteome-constrained genome-scale metabolic model of L. lactis (pcLactis), which explicitly accounts for gene expression processes and associated constraints. Using glucose-limited chemostat data3, we identified the uptake of glucose and arginine as dominant constraints, whose pathway proteins were indeed upregulated in evolved mutants. However, above a growth rate of 0.5 h-1, pcLactis suggests that available enzymes function at their maximum capacity, which allows an increase in growth rate only by altering gene expression to change metabolic fluxes, as was mainly observed for arginine metabolism. Thus, our integrative analysis of flux and proteomics data with a proteome-constrained model is able to identify and explain the constraints that form targets of regulation and fitness improvement in nutrient-rich growth environments.

systems biology↗

bioRxiv2020

Unravelling lactate-acetate conversion to butyrate by intestinal Anaerobutyricum and Anaerostipes species

The D-and L-forms of lactate are important fermentation metabolites produced by intestinal bacteria but have been found to negatively affect mucosal barrier function and human health. Of interest, both enantiomers of lactate can be converted with acetate into the presumed beneficial butyrate by a phylogenetically related group of anaerobes, including Anaerobutyricum and Anaerostipes spp. This is a low energy yielding process with a partially unknown pathway in Anaerobutyricum and Anaerostipes spp. and hence, we sought to address this via a comparative genomics, proteomics and physiology approach. We focused on Anaerobutyricum soehngenii and compared its growth on lactate with that on sucrose and sorbitol. Comparative proteomics revealed a unique active gene cluster that was abundantly expressed when grown on lactate. This active gene cluster, lctABCDEF, encodes a lactate dehydrogenase (lctD), electron transport proteins A and B (lctCB), along with a nickel-dependent racemase (lctE) and a lactate permease (lctF). Extensive search of available genomes of intestinal bacteria revealed this gene cluster to be highly conserved in only Anaerobutyricum and Anaerostipes spp. The present study demonstrates that A. soehngenii and several related Anaerobutyricum and Anaerostipes spp. are highly adapted for a lifestyle involving lactate plus acetate utilization in the human intestinal tract.

microbiology↗