Biology subjects

Kolodkin-Gal, I.

Publications and source records attributed to Kolodkin-Gal, I..

14 recordsLinked to original sources

bioRxiv2026

Achilles Heel: Type VII Toxin/Antitoxin System Hha/TomB Facilitates Phage Infection by Regulating Cryptic Prophage Lytic Genes

Toxin/antitoxin systems (TAs) are stress-response loci most-closely linked to stabilizing mobile genetic elements such as plasmids and inhibiting phage infection. In contrast to the phage inhibition paradigm, here we demonstrate that the type VII TA system, Hha/TomB, facilitates rather than inhibits Escherichia coli infection with 12 BASEL lytic phages (seven Tequatrovirus phages and five Vequintavirus members), including phage 48 (a Vequintavirus), which we chose to focus on due to its clear phenotype. Toxin Hha regulates cryptic prophage lytic genes, and antitoxin TomB masks Hha toxin activity by oxidizing a conserved Hha cysteine residue. We find here that the mechanism by which Hha increases infection is via activation of cryptic prophage lytic genes. Therefore, this is one of the first reports of phages and a type VII TA system as well as the first report of a TA system that is not advantageous for phage inhibition but instead may be an Achilles heel that is manipulated by lytic phages.

molecular biology↗

bioRxiv2026

Deciphering the Acetaldehyde Signaling Network Underlying Bacterial Escape from Protozoan Predators

Entamoeba histolytica (Eh) is a formidable intestinal pathogen, yet the ecological principles governing its invasion of the gut microbiome remain elusive. Upon colonization, Eh encounters resident bacteria typically sequestered within aggregates and biofilms. While Eh utilizes cysteine proteinases to degrade biofilm matrices and access bacterial prey, the strategies bacteria employ to sense and evade this predation are largely unknown. Here, we identify a metabolic signalling axis that allows the probiotic bacterium Bacillus subtilis to perceive and respond to predatory Eh. In the absence of mitochondria, Eh relies on fermentative glycolysis, using alcohol dehydrogenase to produce distinct metabolic byproducts. Using quantitative proteomics and single-cell imaging, we demonstrate that B. subtilis detects the Eh-derived metabolite acetaldehyde as a proxy for predator presence. By mapping the acetaldehyde-responsive regulatory network, we show how this metabolic input is transduced to control the motility machinery, while our data suggest that the broader predatory secretome acts as a multi-modal signal influencing multiple bacterial physiological programs. This chemical cue triggers a rapid phenotypic switch in B. subtilis, driving a transition towards a motile, planktonic "flight" response. Our findings reveal that commensal bacteria exploit the unique metabolic signature of anaerobic parasites to coordinate defensive behaviours, highlighting how inter-kingdom signalling shapes microbiome architecture during infection.

microbiology↗

bioRxiv2026

Assessing soluble and insoluble calcium sources for growth, biofilm formation, and biomineralization in Bacillus subtilis.

Biofilms formed by soil microbes hold immense potential for bioremediation, carbon dioxide sequestration, and the development of sustainable cementitious materials. However, quantifying the complex temporal coupling among bacterial growth, extracellular matrix (ECM) production, and mineralization dynamics remains a significant challenge due to the inherent nonlinearity of these processes and signal noise in high-throughput assays. To address this, we utilized an automated kinetic framework to evaluate the biomineralization competence of Bacillus subtilis under varying calcium regimes. Our results demonstrate that calcium carbonate promotes microbial growth as effectively as soluble calcium acetate, indicating that B. subtilis actively solubilizes the crystalline powder. Despite this growth efficacy, calcium carbonate was an inadequate source for macro-calcite production compared to organic salts. By quantifying expression from the sinI promoter, which initiates SinI production to activate matrix synthesis, we suggested that calcium-acetate-driven extracellular matrix (ECM) expression significantly enhances the structural template required for robust biomineralization. Kinetic expression analysis and consolidation assays indicate that overproduced ECM partially mitigates crystalline calcite growth defects, offering a baseline for utilizing mineral-rich construction waste ImportanceBiomineralization in B. subtilis has been studied as a potential avenue for developing engineered living materials. However, organic calcium salts, such as calcium acetate and calcium lactate, typically yield higher bioconversion efficiencies than certain inorganic sources often used in construction. This advantage has been largely attributed to the metabolic utilization of the organic anion as a carbon source, thereby promoting local alkalinity. Here, our experiments indicate that this limitation is influenced by structural and regulatory constraints rather than purely chemical barriers. Moreover, we explored a targeted synthetic biology approach to address this by genetically decoupling matrix production from native environmental sensing, thereby engineering strains with increased matrix production that can partially overcome the limitations of non-inductive substrates. This approach can contribute to the development of sustainable construction technologies, moving toward actively programmed Engineered Living Materials (ELMs).

microbiology↗

bioRxiv2024

Differential coping strategies exerted by biofilm and planktonic cells of the beneficial bacterium B. subtilis in response to the protozoan predator Entamoeba histolytica.

The human protozoan parasite Entamoeba histolytica causes amebiasis and interacts with both beneficial and harmful members of the microbiome. In previous studies, it was shown that E. histolytica can break down pre-established biofilms of B. subtilis in a time- and dose-dependent manner. Inhibiting parasitic cysteine proteases impairs biofilm degradation. However, it is still unknown whether bacteria can sense this process and respond to the degradation of the biofilms. Here, our research demonstrates a multi-layered response of probiotic bacteria to the parasite, which differs between planktonic bacteria and pre-established biofilms. Sensing the activity of cysteine proteases from E. histolytica, the bacteria activate the general stress response and, to a lesser extent, the cell wall stress response, making the surviving biofilm members more resistant to mild stressors. On the other hand, planktonic cells exposed to the predators lysate deactivate the expression of genes associated with biofilm formation while inducing their motility to avoid predation. Overall, our results indicate that bacteria have evolved to recognize amoeba predators. Furthermore, the partially digested biofilm cells may have unexpected disadvantages over bacteria that did not encounter a predator. These findings may be useful in developing more efficient probiotic strains that are resilient to amoebiasis.

microbiology↗

bioRxiv2024

Biofilm Formation and Niche Adaptation Play Fundamental Role in Shaping Long-Distance Transported Dust Microbial Community

The long-distance transport of microorganisms via dust events is expected to become more prominent and intensified. At the same time, the survival mechanisms of air/dust-borne bacteria and their possible contribution to global processes remain poorly understood. In this study, we characterized Bacillus species from transitional season dust storms, previously identified as a significant component of the bioactive community of the dust microbiome. Our results demonstrated substantial growth and biofilm formation diversification, potentially linked to niche adaptation and surface-associated biofilm formation within heterogeneous dust particles. Most dust isolates form biofilms while exhibiting different preferences for media composition. Sterile dust induced biofilm formation, growth, matrix gene expression of B. subtilis, and robust biofilms in key related dust isolates. Overall, our results highlight the significance of biofilm formation as an adaptive mechanism in the distinct habitat of dust storms, with niche adaptation potentially playing a role in microenvironments within the dust particle. These results hold significant potential for implications on terrestrial and aquatic ecology and health, suggesting a pivotal process by which bacteria survive and evolve in this understudied habitat.

microbiology↗

bioRxiv2023

Metabolic rewiring of the probiotic bacterium Lacticaseibacillus rhamnosus GG contributes to cell wall remodeling and antimicrobials production

Lacticaseibacillus rhamnosus GG (LGG) is a Gram-positive beneficial bacterium that resides in the human intestinal tract and belongs to the family of lactic acid bacteria (LAB). This bacterium is a widely used probiotic and was suggested to provide numerous benefits for human health. However, as in most LAB strains, the molecular mechanisms that mediate the competitiveness of probiotics under different diets remain unknown. Fermentation is a fundamental process in LAB, allowing the oxidation of simple carbohydrates (e.g., glucose, mannose) for energy production under conditions of oxygen limitation, as in the human gut. Our results indicate that fermentation reshapes the metabolome, volatilome, and proteome architecture in LGG. Furthermore, fermentation alters cell envelope remodeling and peptidoglycan biosynthesis, which leads to altered cell wall thickness, aggregation properties, and cell wall composition. In addition, fermentable sugars induced secretion of known and novel metabolites and proteins targeting the enteric pathogens Enterococcus faecalis and Salmonella Enterica serovar Typhimurium. Overall, our results link the common metabolic regulation of cell wall remodeling, aggregation to host tissues, biofilm formation in probiotic strains, and connect the production of antimicrobial effectors with metabolome reprogramming. These findings provide novel insights into the role of nutrition in the establishment of LGG in the gastrointestinal tract.

microbiology↗

bioRxiv2022

Digestive exophagy of Bacterial Biofilms by an Amoeba Predator is Mediated by Specific Biofilm Recognition

The human protozoan parasite Entamoeba histolytica is responsible for amebiasis, a disease endemic to developing countries. E. histolytica trophozoites are released from the cysts to colonize the large intestine, where they primarily feed on bacterial cells. In these scenarios, bacterial cells form aggregates or structured communities too large for phagocytosis. Our results show that E. histolytica can degrade pre-established biofilms of Bacillus subtilis and Escherichia coli in a dose- and time-dependent manner. Surprisingly, trophozoites incubated with B. subtilis biofilm exhibit a unique transcriptome signature compared to those incubated with planktonic cells or without bacteria. Biofilm-induced genes include cysteine proteases (CPs), and the general inhibition of CPs by E64D or by the use of specific small-RNA (sRNA)-based RNA interference impairs the degradation of biofilms by E. histolytica. The degradation of B. subtilis extracellular matrix (ECM) protein TasA by CPs is associated with partial biofilm digestion and activation of the stress response in the interacting B. subtilis cells. The interaction with B. subtilis biofilms was also associated with lower levels of oxidoreductases. Oxidoreductase downregulation can be a readout of the embedding of E. histolytica trophozoites within the biofilm-produced extracellular matrix, reducing their exposure to oxidative stress (OS). Our results indicate that parasites may digest biofilms by a controlled mechanism of digestive exophagy as secretion of digestive enzymes as a conserved mechanism for biofilm degradation allows phagocytic digestion of biofilm cells. Furthermore, the partially digested biofilms can serve as an unexpected shield protecting parasites from oxidative environments and thereby may regulate the persistence and virulence of the parasite.

microbiology↗

bioRxiv2022

Context-dependent differences in the functional responses of Lactobacillaeca strains to fermentable sugars

Lactobacillaceae are Gram-positive rods, facultative anaerobes, and belong to the lactic acid bacteria (LAB) that frequently serve as probiotics. We systematically compared five LAB strains for the effects of different carbohydrates on their free-living and biofilm lifestyles. We found that fermentable sugars triggered a heterogeneous response in LAB strains, frequently manifested specifically in altered carrying capacity during planktonic growth and colony development. The fermentation capacities of the strains were compatible and could not account for heterogeneity in their differential carrying capacity in liquid and on a solid medium. Among tested LAB strains, L. paracasei, and L. rhamanosus GG survived self-imposed acid stress while L. acidophilus was extremely sensitive to its own glucose utilization acidic products. The addition of a buffering system during growth on a solid medium significantly improved the survival of most tested probiotic strains during fermentation. We suggest that the optimal performance of the beneficial microbiota members belonging to lactobacilli is heterogeneous and varies as a function of the growth model and the dependency on a buffering system.

microbiology↗

bioRxiv2022

Arabidopsis thaliana induces multigenerational stress tolerance against biotic and abiotic stressors and memorization of host colonization in Bacillus subtilis

Bacillus subtilis is a beneficial bacterium that supports plant growth and protects it from bacterial, fungal, and viral infections. Here using a simplified system of B. subtilis, and Arabidopsis thaliana interactions, we found that history-dependent behavior is a potentially important manifestation of host colonization, worth classifying and quantifying. To study history-dependent adaptation to plant hosts, we develop a simple framework for measuring the physiological memory of B. subtilis following its interaction with Arabidopsis thaliana. We found that A. thaliana secretions reduce the lag time in pre-exposed bacteria compared with naive B. subtilis cells, even after their complete removal. Pre-exposed B. subtilis cells colonized plant roots more efficiently than naive bacteria, and were more resistant to biotic and abiotic stressors such as salicylic acid, and high salinity. Descendants of bacteria treated with plant secretions had an advantage in the competition against unexposed bacteria for root colonization. The effect of plant secretions was independent of their roles as nitrogen and carbon sources. Transcriptome analysis of both ancestors and descendants revealed that a specific set of plant-induced processes, among them c-di-AMP homeostasis, and the general stress response, maintain the signature of association with the plant in descendants of pre-exposed bacteria. Consistently, plant secretions compensated for the loss of c-di-AMP cyclases but required the general stress response and the master regulator Spo0A to exert their short and long-term effects. Overall, our work demonstrates that bacterial memory manifested by multigenerational reversible adaptation to plant hosts confirms an advantage to symbiotic bacteria during competition.

microbiology↗

bioRxiv2021

Imaging Flow Cytometry reveals a dual role for exopolysaccharides in biofilms: To promote self-adhesion while repelling non-self-community members

In nature, bacteria are establishing differentiated communities referred to as biofilms. These multicellular communities are held together by self-produced polymers that allow the community members to adhere to the surface as well as to neighbor bacteria. Here, we report that exopolysaccharides prevent Bacillus subtilis from co-aggregating with a distantly related bacterium Bacillus mycoides, while maintaining their role in promoting self-adhesion and co-adhesion with phylogenetically related bacterium, Bacillus atrophaeus. The defensive role of the exopolysaccharides is due to the specific regulation of bacillaene. Single cell analysis of biofilm and free-living bacterial cells using imaging flow cytometry confirmed a specific role for the exopolysaccharides in microbial competition repelling B. mycoides. Unlike exopolysaccharides, the matrix protein TasA induced bacillaene but inhibited the expression of the biosynthetic clusters for surfactin, and therefore its overall effect on microbial competition during floating biofilm formation was neutral. Thus, the exopolysaccharides provide a dual fitness advantage for biofilm-forming cells, as it acts to promote co-aggregation of related species, as well as, a secreted cue for chemical interference with non-compatible partners. These results experimentally demonstrate a general assembly principle of complex communities and provides an appealing explanation for how closely related species are favored during community assembly. Furthermore, the differential regulation of surfactin and bacillaene by the extracellular matrix may explain the spatio-temporal gradients of antibiotic production within biofilms.

microbiology↗

bioRxiv2021

Quantifying the effects of the plant root on antibiotic production in the beneficial bacterium B. subtilis

Beneficial and probiotic bacteria play an important role in conferring the immunity of their hosts against a wide range of bacterial, viral and fungal diseases. B. subtilis is a bacterium that protects the plant from various pathogens due to its capacity to produce an extensive repertoire of antibiotics. At the same time, the plant microbiome is a highly competitive niche, with multiple microbial species competing for space and resources, a competition that can be determined by the antagonistic potential of each microbiome member. Therefore, regulating antibiotic production in the rhizosphere is of great significance to eliminate pathogens and to establish beneficial host-associated communities. In this work, we used Bacillus subtilis as a model to investigate the role of plant colonization in antibiotic production. Flow cytometry and Image-stream cytometry analysis supported the notion that A. thaliana specifically induced the transcription of the biosynthetic clusters for the non-ribosomal peptides surfactin, bacilysin and plipastatin and the polyketide bacillaene. This induction could be beneficial for the root as all clusters were shown to antagonize plant pathogens. Consistently, the root failed to induce PenP, a {beta}-lactamase that increases only the fitness of the bacteria. Our results can be translated to improve the performance and competitiveness of beneficial members of the plant microbiome.

microbiology↗

bioRxiv2021

Resolving the conflict between antibiotic production and rapid growth by recognition of peptidoglycan of susceptible competitors

Microbial communities employ a variety of complex strategies to compete successfully against competitors sharing their niche, with antibiotic production being a common strategy of aggression. Here, by systematic evaluation of all non-ribosomal peptides (NRP) produced by B. subtilis clade, we revealed that they acted either synergistically or additively to effectively eliminate phylogenetically distinct competitors. All four major NRP biosynthetic clusters were also imperative for the survival of B. subtilis in a complex community extracted from the rhizosphere. The production of NRP came with a fitness cost manifested in growth inhibition, rendering NRP synthesis uneconomical when growing in proximity to a phylogenetically close species, carrying resistance against the same antibiotics. To resolve this conflict and ease the fitness cost, NRP production was only induced by the presence of peptidoglycan cue from a sensitive competitor. These results experimentally demonstrate a general ecological concept - closely related communities ("self") are favoured during competition, due to compatibility in attack and defence mechanisms.

microbiology↗

bioRxiv2020

A novel calcium-concentrating compartment drives biofilm formation and persistent infections

In nature, bacteria reside in biofilms - multicellular differentiated communities held together by extracellular matrix. In this work, we identified a novel subpopulation essential for biofilm formation - mineral-forming cells. This subpopulation contains an intracellular calcium-accumulating niche, in which the formation of a calcium carbonate mineral is initiated. As the biofilm colony develops, this mineral grows in a controlled manner, forming a functional macrostructure that serves the entire community. The molecular mechanisms promoting calcite scaffold formation were conserved between three distant phyla - the Gram-positive Bacillus subtilis, Gram-negative Pseudomonas aeruginosa and the actinobacterium Mycobacterium abscessus. Biofilm development of all three species was similarly impaired by inhibition of calcium uptake and carbonate accumulation. Moreover, chemical inhibition and mutations targeting mineralization both significantly reduced the attachment of P. aeruginosa to the lung, as well as the subsequent damage inflicted by biofilms to lung tissues, and restored their sensitivity to antibiotics. The evolutionary conserved cellular pathway controlling the fundamental feature of biofilm development uncovered in this work offers novel druggable targets for antibiotics to combat otherwise untreatable biofilm infections.

microbiology↗

bioRxiv2018

A matrix protein acts as a cue to preserve collective motility of B. subtilis biofilm cells

Bacteria in nature are usually found in complex multicellular communities, termed biofilms. Biofilms are generally seen as sessile structures, resulting from downregulation of motility. However, during interspecies competition and predation, biofilm cells were shown to migrate towards competitor colonies. Here, we show that a specific extracellular matrix (ECM) protein, TasA, is essential for collective migration toward potential competitors and serves as a developmental cue that increases the formation of motile offspring from sessile chains. We reveal an effective strategy to maintain migration capacities in bacterial biofilms: besides providing a three-dimensional adhesive scaffold for the cells, TasA acts as a signal within the bacterial community.

microbiology↗