Biology subjects

Hamoen, L. W.

Publications and source records attributed to Hamoen, L. W..

13 recordsLinked to original sources

bioRxiv2026

Transposon library anomaly reveals importance of cell wall teichoic acids for kin discrimination

Genome-wide transposon insertion sequencing (Tn-seq) is a powerful tool to measure the importance of genes for growth. In this study, we applied Tn-seq to the Gram-positive model system Bacillus subtilis, and found that after growth in liquid medium the transposon library lacked transposon insertions in several genes related to lipoteichoic acid biosynthesis and cell wall teichoic acid modification. This was unexpected since these genes are not essential for normal growth. By growing the transposon library as a confluent layer of cells, and as discrete colonies, we found that these genes are only important when the transposon library is grown as a confluent layer. Thus, growing the transposon library as a mixed population reduces the fitness of teichoic acid mutants. We provide further evidence showing that this anomaly is caused by the production of multiple autologous antimicrobials and/or toxins in the growth medium. These findings show that growing genome-wide mutant libraries as mixed cultures can influence the mutant library composition.

microbiology↗

bioRxiv2026

A fungal pathogen effector that shapes host plant microbiota kills bacteria through lipoteichoic acid binding and membrane disruption

Antimicrobial proteins are ancient and widespread molecules that contribute to survival across all domains of life. We previously showed that fungal plant pathogens secrete antimicrobial proteins to suppress antagonistic members of the host microbiota and thereby promote host colonization. The soil-borne plant pathogen Verticillium dahliae employs the bactericidal protein VdAve1 for this purpose. Here, we elucidate the mode of action of VdAve1 and define its mechanism as a distinct type of antimicrobial activity. Nuclear magnetic resonance (NMR) analysis revealed that VdAve1 adopts a jelly-roll barrel-like fold. Functionally, VdAve1 disrupts bacterial plasma membranes, and synthetic peptides derived from its positively charged regions retain antimicrobial activity, indicating that these regions contribute to its function. We further show that the bacterial model system Bacillus subtilis responds to VdAve1 by modifying teichoic acids, and that loss of these modifications increases bacterial sensitivity. Consistent with this, VdAve1 binds lipoteichoic acid (LTA), a major component of the Gram-positive cell wall. Together, our findings support a model in which VdAve1 binds LTA to localize at the bacterial surface, where it perturbs the plasma membrane, leading to membrane collapse and cell death. SIGNIFICANCE STATEMENTPlant-pathogenic microbes secrete effector proteins to promote host colonization. While these proteins are typically studied for their roles in host manipulation, some affect host-associated microbial communities by exhibiting antimicrobial activity. However, their modes of action remain largely unexplored. The fungal plant pathogen Verticillium dahliae secretes the antimicrobial effector VdAve1 to inhibit bacterial antagonists in the host microbiota. Here, we characterize the mode of action of VdAve1. We show that VdAve1 binds lipoteichoic acid, a major component of the cell wall of Gram-positive bacteria. This promotes VdAve1 accumulation close to the plasma membrane, and through electrostatic interactions it disrupts the plasma membrane. We further determine VdAve1 structure by NMR, revealing VdAve1 adopts a {beta}-barrel-like fold. Together, our findings show the mechanism by which a fungal effector inhibits bacterial growth and thereby how it may influence host-associated bacterial communities.

microbiology↗

bioRxiv2026

Tetrabromobisphenol A (TBBPA) inhibits Bacillus subtilis as a Membrane Active Antibacterial Agent

Antimicrobial resistance (AMR) is a pressing global public health crisis, necessitating novel antimicrobial agents and mechanistic insights. Tetrabromobisphenol A (TBBPA), a widely used brominated flame retardant, exhibits potent activity against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) without inducing resistance, yet its mode of action remains unclear. Using Bacillus subtilis as a model, we investigated TBBPAs antibacterial mechanism via extensive bacterial cytological profiling, fluorescence imaging, and mutant validation. We found that TBBPA causes membrane depolarization and disruption, based on Thioflavin T release, MinD mislocalization, FM5-95 fluorescence aggregation, and propidium iodide penetration. In fact, TBBPA can destabilizes giant unilamellar lipid vesicles in vitro. The induced membrane damage triggers several downstream effects, including MreB immobilization, which impairs cell wall synthesis, inhibition of DNA replication and translation, and increased autolysin activation leading to cell lysis. In conclusion, this study suggests that TBBPA directly targets the cell membrane, causing disruption of multiple essential processes, and leads to activation of autolysins, resulting in lysis. These findings highlight TBBPAs prospective utility as an anti-Gram-positive agent; nevertheless, concerns over its potential side effects necessitate further investigations into its safety profile prior to clinical translation.

microbiology↗

bioRxiv2024

Cyclical MinD membrane affinity differences are not necessary for MinD gradient formation in Bacillus subtilis

Proteins can diffuse micrometers in seconds, yet bacterial cells are able to maintain stable protein gradients. The best studied bacterial protein gradient is the Min system of Escherichia coli. In rod-shaped bacteria the MinCD proteins prevent formation of minicells by inhibiting FtsZ polymerization close to the cell poles. In E. coli these proteins oscillate between cell poles within a minute, resulting in an increased MinCD concentration at the poles. This oscillation is caused by the interaction between MinD and the protein MinE, which form an ATP-driven reaction-diffusion system, whereby the ATPase MinD cycles between a monomeric cytosolic and a dimeric membrane attached states. Bacillus subtilis also has MinCD, but lacks MinE. In this case MinCD form a static gradient that requires the transmembrane protein MinJ, located at cell poles and cell division sites. A recent reaction-diffusion model was successful in recreating the MinD gradient in B. subtilis, assuming that MinD cycles between cytosol and membrane, like in E. coli. Here we show that the monomeric and dimeric states of B. subtilis MinD have comparable membrane affinities, that MinD interacts with MinJ as a dimer, and that MinJ is not required for membrane localization of MinD. Based on these new findings we tested different models, using kinetic Monte Carlo simulations, and found that a difference in diffusion rate between the monomer and dimer, rather than a difference in membrane affinity, is important for B. subtilis MinCD gradient formation.

microbiology↗

bioRxiv2024

Divisome minimization shows that FtsZ and SepF can form an active Z-ring, and reveals BraB as a new cell division influencing protein in Bacillus subtilis

One of the aims of synthetic biology is the construction of a minimal cell. Since bacteria are the simplest life forms, they are the preferred blueprint for such a cell. In this study, we tried to find the minimal set of proteins required for division of a cell wall containing cell, using the bacterial model system Bacillus subtilis. Bacterial cytokinesis begins with polymerization of the tubulin homologue FtsZ at midcell into the Z-ring, which recruits the late cell division protein that synthesize the division septum. Assembly of FtsZ is carefully regulated, involving a dozen conserved cell division proteins. These proteins are not essential, but removing more than one is in many cases lethal. We made use of known suppressor mutations to find a gene deletion route that eventually enabled us to remove eight conserved cell division proteins: ZapA, MinC, MinJ, UgtP, ClpX, Noc, EzrA and FtsA. Only FtsZ and its membrane anchor SepF appeared to be required for Z-ring formation. Interestingly, SepF is also the FtsZ anchor in archaea, and both proteins may date back to the Last Universal Common Ancestor (LUCA). Curiously, viability was not greatly affected by the multiple deletions, although the frequency of cell division was considerably reduced. However, genome sequencing exposed the accumulation of several suppressor mutations, and revealed an unexpected cell division regulation function for the branched chain amino acid transporter BraB. The implications of these findings for the role of SepF in cell division, and the construction of a minimal cell division machinery are discussed.

microbiology↗

bioRxiv2022

The conserved protein WhiA influences branched-chain fatty acid precursors in Bacillus subtilis

The conserved WhiA protein family is present in most Gram-positive bacteria and plays a role in cell division. WhiA contains a DNA-binding motive and has been identified as a transcription factor in actinomycetes. In Bacillus subtilis, the absence of WhiA influences cell division and chromosome segregation, however, it is still unclear how WhiA influences these processes, but the protein does not seem to function as transcription factor in this organism. To further investigate the function of WhiA in B. subtilis, we performed a yeast two-hybrid screen to find interaction partners, and a Hi-C experiment to reveal possible changes in chromosome conformation. The latter experiment indicated a reduction in short range chromosome interactions, but how this would affect either cell division or chromosome segregation is unclear. Based on adjacent genes, a role in carbon metabolism was put forward. To study this, we measured exometabolome fluxes during growth on different carbon sources. This revealed that in {Delta}whiA cells the pool of branched-chain fatty acid precursors is lower. However, the effect on the membrane fatty acid composition was minimal. Transcriptome data could not link the metabolome effects to gene regulatory differences. IMPORTANCEWhiA is a conserved DNA binding protein that influences cell division and chromosome segregation in the Gram-positive model bacterium B. subtilis. The molecular function of WhiA is still unclear, but a previous study has suggested that the protein does not function as a transcription factor. In this study, we used yeast two-hybrid screening, chromosome conformation capture analysis, metabolomics, transcriptomics and fatty acid analysis to obtain more information about the workings of this enigmatic protein.

microbiology↗

bioRxiv2022

Transcriptome analyses using regulon, functional category, and operon information with GINtool

When analysing transcriptome data, threshold values are chosen to decide whether the regulation of a gene is relevant or not, however this may result in the loss of valuable information. To overcome this problem it can be useful to analyse regulons instead of individual genes, to harness the statistical power of combining genes. Another advantage of a regulon-based analysis is that it provides direct insights into the activity of regulatory pathways, which is the essence of transcriptome analyses. We have developed a software tool called GINtool that can use regulon information to analyse transcriptome data. GINtool includes the option to take the activity mode of a regulator in account, which is important when a regulator can function both as an activator and repressor. GINtool also contains two novel graphical representations that greatly facilitate the visual inspection of regulon-based transcriptome analyses. Additional features of GINtool includes the evaluation of transcriptome data using functional categories, and the analysis of gene expression differences within operons. To ease the analyses and downstream processing of figures, GINtool has been developed as an add-in for Excel.

bioinformatics↗

bioRxiv2021

Classification of antimicrobial mechanism of action using bacterial time-lapse imaging

Antimicrobial resistance is a major threat to human health. Basic knowledge of antimicrobial mechanism of action (MoA) is imperative for patient care and for identification of novel antimicrobials. However, the process of antimicrobial MoA identification is relatively laborious. Here, we developed a simple, quantitative time-lapse fluorescence imaging method, Dynamic Bacterial Morphology Imaging (DBMI), to facilitate this process. It uses a membrane dye and a nucleoid dye to track the morphological changes of single Bacillus subtilis cells in response to antimicrobials for up to 60 min. DBMI of bacterial cells facilitated assignment of the MoAs of 14 distinct, known antimicrobial compounds to the five main classes. Using this method, we found that the poorly studied antimicrobial, harzianic acid, a secondary metabolite that we purified from the fungal culture of Oidiodendron flavum, targets the cell envelope. We conclude that DBMI is a simple method, which facilitates rapid classification of the MoA of antimicrobials in functionally distinct classes.

microbiology↗

bioRxiv2021

Different resource allocation in a Bacillus subtilis population displaying bimodal motility

To cope with sudden changes in their environment, bacteria can use a bet-hedging strategy by dividing the population into cells with different properties. This so-called bimodal or bistable cellular differentiation is generally controlled by positive feedback regulation of transcriptional activators. Due to the continuous increase in cell volume, it is difficult for these activators to reach an activation threshold concentration when cells are growing exponentially. This is one reason why bimodal differentiation is primarily observed from the onset of the stationary phase when exponential growth ceases. An exception is the bimodal induction of motility in Bacillus subtilis, which occurs early during exponential growth. Several mechanisms have been put forward to explain this, including double negative-feedback regulation and the stability of the mRNA molecules involved. In this study, we used fluorescence-assisted cell sorting to compare the transcriptome of motile and non-motile cells and noted that expression of ribosomal genes is lower in motile cells. This was confirmed using an unstable GFP reporter fused to the strong ribosomal rpsD promoter. We propose that the reduction in ribosomal gene expression in motile cells is the result of a diversion of cellular resources to the synthesis of the chemotaxis and motility systems. In agreement, single-cell microscopic analysis showed that motile cells are slightly shorter than non-motile cells, an indication of slower growth. We speculate that this growth rate reduction can contribute to the bimodal induction of motility during exponential growth. IMPORTANCETo cope with sudden environmental changes, bacteria can use a bet-hedging strategy and generate different types of cells within a population, so called bimodal differentiation. For example, a Bacillus subtilis culture can contain both motile and non-motile cells. In this study we compared the gene expression between motile and non-motile cells. It appeared that motile cells express less ribosomes. To confirm this, we constructed a ribosomal promoter fusion that enabled us to measure expression of this promoter in individual cells. This reporter fusion confirmed our initial finding. The re-allocation of cellular resources from ribosome synthesis towards synthesis of the motility apparatus results in a reduction in growth. Interestingly, this growth reduction has been shown to stimulate bimodal differentiation.

microbiology↗

bioRxiv2019

New flat embedding method for transmission electron microscopy reveals an unknown mechanism of tetracycline

Transmission electron microscopy (TEM) is an important imaging technique in bacterial research and requires ultrathin sectioning of resin embedding of cell pellets. This method consumes milli- to deciliters of culture and results in sections of randomly orientated cells. For rod-shaped bacteria, this makes it exceedingly difficult to find longitudinally cut cells, which precludes large-scale quantification of morphological phenotypes. Here, we describe a new fixation method using either thin agarose layers or carbon-coated glass surfaces that enables flat embedding of bacteria. This technique allows for the observation of thousands of longitudinally cut rod-shaped cells per single section and requires only microliter culture volumes. We successfully applied this technique to Gram-positive Bacillus subtilis, Gram-negative Escherichia coli, the tuberculosis vaccine strain Mycobacterium bovis BCG, and the cell wall-lacking mycoplasma Acholeplasma laidlawii. To assess the potential of the technique to quantify morphological phenotypes, we examined cellular changes induced by a panel of different antibiotics. Surprisingly, we found that the ribosome inhibitor tetracycline causes significant deformations of the cell membrane. Further investigations showed that the presence of tetracycline in the cell membrane changes membrane organization and affects the peripheral membrane proteins MinD, MinC, and MreB, which are important for regulation of cell division and elongation. Importantly, we could show that this effect is not the result of ribosome inhibition but is a secondary antibacterial activity of tetracycline that has defied discovery for more than 50 years.\n\nSignificanceBacterial antibiotic resistance is a serious public health problem and novel antibiotics are urgently needed. Before a new antibiotic can be brought to the clinic, its antibacterial mechanism needs to be elucidated. Transmission electron microscopy is an important tool to investigate these mechanisms. We developed a flat embedding method that enables examination of many more bacterial cells than classical protocols, enabling large-scale quantification of phenotypic changes. Flat embedding can be adapted to most growth conditions and microbial species and can be employed in a wide variety of microbiological research fields. Using this technique, we show that even well-established antibiotics like tetracycline can have unknown additional antibacterial activities, demonstrating how flat embedding can contribute to finding new antibiotic mechanisms.

microbiology↗

bioRxiv2019

Control of septum thickness by a large protein ring

Gram-positive bacteria divide by forming a thick cross wall. How the thickness of this septal wall is controlled is unknown. In this type of bacteria, the key cell division protein FtsZ is anchored to the cell membrane by two proteins, FtsA and SepF. We have isolated SepF homologues from different bacterial species and found that they all polymerize into large protein rings with diameters varying from 19 to 41 nm. Importantly, these values correlated well with the thickness of their septa. To test whether ring diameter determines septal thickness, we tried to construct different SepF chimeras with the purpose to manipulate the diameter of the SepF protein ring. This was indeed possible and confirmed that the conserved core domain of SepF determines ring diameter. Importantly, when SepF chimeras with a smaller diameter were expressed in the bacterial host Bacillus subtilis, the thickness of its septa also became smaller. These results strongly support a model in which septal thickness is controlled by curved molecular clamps formed by SepF polymers attached to the leading edge of nascent septa. This also implies that the intrinsic shape of a protein polymer can function as a mould to shape the cell wall. Significance StatementMany bacteria form a thick cell wall and divide by forming a cross wall. How they control the thickness of their cell wall and cross wall is unknown. In this study we show that in these bacteria the cell division protein SepF forms very large protein rings with diameters that correspond to the diameter of their cross walls. Importantly, when we reduced the diameter of SepF rings in the bacterial host Bacillus subtilis the cross wall also became thinner. These results provide strong evidence that a large protein ring can function as a mould to control the thickness of the cell wall that divides these bacterial cells.

microbiology↗

bioRxiv2017

Membrane curvature and the Tol-Pal complex determine polar localization of the chemoreceptor Tar in E. coli

Chemoreceptors are localized at the cell poles of Escherichia coli and other rod-shaped bacteria. Over the years different mechanisms have been put forward to explain this polar localization; from stochastic clustering, membrane curvature driven localization, interactions with the Tol-Pal complex, to nucleoid exclusion. To evaluate these mechanisms, we monitored the cellular localization of the aspartate chemoreceptor Tar in different deletion mutants. We did not find any indication for either stochastic cluster formation or nucleoid exclusion. However, the presence of a functional Tol-Pal complex appeared to be essential to retain Tar at cell poles. This finding also implies that the curvature of cell poles does not attract chemoreceptor complexes. Interestingly, Tar still accumulated at midcell in tol and in pal deletion mutants. In these mutants, the protein appears to gather at the base of division septa, a region characterised by strong membrane curvature. Chemoreceptors, like Tar, form trimer-of-dimers that bend the cell membrane due to a rigid tripod structure with an estimated curvature of approximately 37 nm. This curvature approaches the curvature of the cell membrane generated during cell division, and localization of chemoreceptor tripods at curved membrane areas is therefore energetically favourable as it lowers membrane tension. Indeed, when we introduced mutations in Tar that abolish the rigid tripod structure, the protein was no longer able to accumulate at midcell or cell poles. These findings favour a model where chemoreceptor localization in E. coli is driven by strong membrane curvature and association with the Tol-Pal complex.\n\nImportanceBacteria have exquisite mechanisms to sense and to adapt to the environment they live in. One such mechanism involves the chemotaxis signal transduction pathway, in which chemoreceptors specifically bind certain attracting or repelling molecules and transduce the signals to the cell. In different rod-shaped bacteria, these chemoreceptors localize specifically to cell poles. Here, we examined the polar localization of the aspartate chemoreceptor Tar in E. coli, and found that membrane curvature at cell division sites and interaction with the Tal-pol protein complex, localize Tar at cell division sites, the future cell poles. This study shows how membrane curvature can guide localization of proteins in a cell.

microbiology↗

bioRxiv2016

In culture cross-linking of bacterial cells reveals proteome scale dynamic protein-protein interactions at peptide level

Identification of dynamic protein-protein interactions at the peptide level on a proteomic scale is a challenging approach that is still in its infancy. We have developed a system to cross-link cells directly in culture with the special lysine cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). We used the Gram positive model bacterium Bacillus subtilis as an exemplar system. Within 5 min extensive intracellular cross-linking was detected, while intracellular cross-linking in a Gram-negative species, Escherichia coli, was still undetectable after 30 min, in agreement with the low permeability in this organism for lipophilic compounds like BAMG. We were able to identify 82 unique inter-protein cross-linked peptides with less than a 1% false discovery rate by mass spectrometry and genome-wide data base searching. Nearly 60% of the inter-protein cross-links occur in assemblies involved in transcription and translation. Several of these interactions are new, and we identified a binding site between the {delta} and {beta}' subunit of RNA polymerase close to the downstream DNA channel, providing a clue into how {delta} might regulate promoter selectivity and promote RNA polymerase recycling. Our methodology opens new avenues to investigate the functional dynamic organization of complex protein assemblies involved in bacterial growth.

biochemistry↗