Biology subjects

Bernal, P.

Publications and source records attributed to Bernal, P..

9 recordsLinked to original sources

bioRxiv2026

Rsm-mediated post-translational control of the Pseudomonas putida Type VI Secretion System

The Type VI secretion system (T6SS) is a bacterial nanoweapon that injects toxic effectors into prokaryotic and eukaryotic cells. It is widely found among gram-negative bacteria and provides a significant fitness advantage in interbacterial competition. Pseudomonas putida KT2440 possesses three T6SS clusters (K1-, K2- and K3-T6SS) that combat phytopathogens. This makes this strain a potent biocontrol agent that protects plants from pathogens and can be further enhanced by a better understanding of its T6SS regulation. Although the core components of T6SS are conserved, the elements controlling its regulation differ among bacterial species. T6SS activity is regulated by various factors acting at different levels, from transcription to post-translational modification, to ensure precise control of its activity. Here, we demonstrate the critical importance that the three Rsm proteins, RsmIEA, have in controlling the K1-T6SS structural components and related orphan elements at the post-transcriptional level in Pseudomonas putida. We identified multiple Rsm-binding sites responsible for directly repressing the translation of T6SS proteins (Hcp1 and Hcp5) and their associated effectors (Tke2 and Tke7). Derepression of K1-T6SS mRNA in the rsmIEA mutant led to enhanced translation and expression of the K1-T6SS components and effectors, and critically increased the number of cells in the population with assembled T6SS. This results in a greater capacity to secrete toxins and kill prey cells via the T6SS-dependent mechanism. Finally, we demonstrate the K1-T6SS ability to kill environmental pathogens, including Salmonella enterica and Erwinia amylovora.

microbiology↗

bioRxiv2026

Spatial control of light-responsive proteins and optogenetics within hydrogels via volumetric bioprinting

Spatiotemporal control over cell fate and behaviour within bioprinted constructs remains a key challenge in tissue engineering. Optogenetics offers versatile potential for non-invasive regulation of biological processes. Yet, its integration within large-scale, cell-laden bioprinted materials is still limited, especially considering spatial constraints of existing light delivery methods. In this study, we introduce a novel approach that repurposes tomographic volumetric bioprinting to enable post-printing stimulation of photosensitive protein-switches and optogenetic circuits in cells deep within hydrogel constructs. By converging different bioprinting approaches, computer vision, context-aware model generation, and synthetic biology and cell engineering, we demonstrated selective activation of a fluorescent, light-responsive protein probe within multi-material centimeter-scale constructs. Moreover, leveraging a multi-wavelength volumetric bioprinter, we further demonstrate this concept by selectively stimulating cells expressing a near-infrared optogenetic system that triggers gene expression and the induction of pancreas-specific transcription factors. The described methods provide platforms for remote, repeatable, and localized control of biological events in volumetric constructs, opening new possibilities for advanced tissue models, and dynamic tuning of cell-mediated protein production in engineered living systems.

bioengineering↗

bioRxiv2025

The Pseudomonas putida Type VI Secretion Systems Shape the Tomato Rhizosphere Microbiota

Bacterial competition mechanisms drive microbial community dynamics across diverse ecological niches. The Type VI Secretion System (T6SS) represents a sophisticated nanomachine used by Gram-negative bacteria for contact-dependent elimination of competitors through the delivery of toxic effectors. While the T6SS has been well-documented in mammalian gut microbiota development, its role in shaping plant rhizosphere communities remains poorly understood despite the ecological importance of rhizosphere microbiota. This study investigates how the three Pseudomonas putida KT2440 T6SS clusters influence the tomato rhizosphere microbiota in agricultural soil. Through comprehensive in vitro and in vivo analyses, we demonstrate that while the K2/K3-T6SSs remain inactive under standard laboratory conditions, they become specifically activated in the presence of plant pathogens, suggesting an adaptive response to competitive pressure. Our experiments with T6SS-deficient mutants reveal that the P. putida T6SSs are essential for effective rhizosphere colonisation, with mutant strains showing significantly reduced colonisation capabilities compared to wildtype strain in competitive soil environments. Most importantly, our data establish that the P. putida T6SSs directly shape the taxonomic diversity and community structure of the rhizosphere microbiota of tomato plants. These results place the T6SS as a critical factor driving the evolution of complex polymicrobial communities within the plant rhizosphere, paralleling its established role in the gut microbiota. This research advances our understanding of the ecological functions of the different T6SSs in P. putida and the molecular mechanisms underlying microbial community assembly in the rhizosphere. Thus, it offers valuable insights for agricultural applications involving beneficial microbes and plant health management strategies.

microbiology↗

bioRxiv2025

The cryo-EM structure of an adaptor-effector complex reveals the mechanism of a widespread pore-forming toxin family

Pseudomonas putida KT2440 is a plant-beneficial rhizobacterium that encodes multiple Type VI secretion systems (T6SS) to outcompete phytopathogens in the rhizosphere. Among its antibacterial effectors, Tke5 has been identified as a potent pore-forming toxin that disrupts ion homeostasis without causing considerable membrane damage. Tke5 belongs to the BTH_I2691 protein family and harbours an N-terminal marker for the type six secretion system effectors (MIX) motif, previously shown to be required for T6SS-dependent secretion in other systems. Many MIX-containing effectors require T6SS adaptor proteins (Tap) for secretion, but until now, the molecular mechanism for adaptor-effector binding has remained elusive. Here, we report the 2.8 [A] cryo-EM structure of the Tap3-Tke5 complex, providing structural and functional insight into how this effector is recruited by its cognate adaptor protein Tap3. Functional dissection shows that the -helical region of Tke5 is sufficient to kill intoxicated bacteria, while its {beta}-rich region likely contributes to target membrane specificity. These findings suggest a general mechanism of MIX-containing BTH_I2691 proteins for Tap recruitment and toxin activity, contributing to our fundamental understanding of a widespread yet understudied toxin family.

microbiology↗

bioRxiv2025

Tke5 is a novel Pseudomonas putida toxin that depolarises membranes killing plant pathogens

The soil bacterium Pseudomonas putida injects toxic proteins into neighbouring competitors, including resilient phytopathogens, using the Type VI secretion system (T6SS). The secretion of toxins endows P. putida with a significant fitness advantage, allowing this biocontrol agent to thrive in plant-related polymicrobial environments and prevent phytopathogen infections. Despite its agricultural significance, the toxin repertoire of P. putida, particularly those secreted via the K2- and K3-T6SSs, remains poorly understood. We present the first comprehensive molecular study of Tke5, a potent toxin encoded within the K3-T6SS, which represents the first comprehensive functional analysis of the BTH_I2691 protein family. Our biophysical data demonstrate that Tke5 is a pore-forming toxin that disrupts bacterial membranes through selective ion transport, inducing membrane depolarisation and cell death. Unlike conventional detergent-like pore-forming toxins, Tke5 preserves overall membrane integrity, avoiding large, non-specific disruptions. This unique mechanism offers a powerful approach to targeting resilient phytopathogens. This study reveals a previously undescribed mode of action within a widespread yet understudied toxin family. Our findings highlight the potential of P. putida as a biocontrol agent, offering alternatives to chemical pesticides by exploiting novel toxin mechanisms. Understanding these bacterial toxins is crucial for developing effective strategies to combat plant pathogens.

microbiology↗

bioRxiv2023

The type VI secretion system of Sinorhizobium fredii USDA257 is required for a successful symbiosis with Glycine max cv Pekin

For agriculture, the symbiosis carried out by rhizobia with legumes stands out as crucial for both economic and environmental reasons. In this process, the bacteria colonize the roots of the plants, inducing the formation of plant organs called nodules. Within these structures, rhizobia fix the environmental nitrogen into ammonia reducing the demand for this essential element required for plant growth. Various bacterial secretion systems (TXSS, Type X Secretion System) are involved in the establishment of this symbiosis, with the T3SS being the most extensively studied. The T6SS is a nanoweapon present in 25% of gram-negative bacteria, commonly used against other gram-negative bacteria, though some of them use it to manipulate eukaryotic cells. Interestingly, although T6SS is widely distributed among rhizobia, whether it has a specific role in symbiosis with legumes remains elusive. Sinorhizobium fredii USDA257 is a fast-growing rhizobium with the capacity to nodulate a great variety of legume plants. This strain harbors a single T6SS cluster, containing the genes encoding all the structural components of the system and two genes encoding potential effectors that could target the cell wall of the plants and/or be acting as a toxin/antitoxin system. We have demonstrated that this system is active and can be induced in poor culture media. In addition, we have seen by fluorescence microscopy that the T6SS is active in nodules. Competition assays between USDA257 and different preys have shown that USDA257 cannot kill any of them using its T6SS under tested conditions. By constract, nodulation assays demonstrated that USDA257 utilizes this protein secretion system to enhance nodulation and competitiveness with its host Glycine max cv Pekin.

microbiology↗

bioRxiv2023

Antibiotic potentiation and inhibition of cross-resistance in pathogens associated with cystic fibrosis

Critical Gram-negative pathogens, like Pseudomonas, Stenotrophomonas and Burkholderia, have become resistant to most antibiotics. Complex resistance profiles together with synergistic interactions between these organisms increase the likelihood of treatment failure in distinct infection settings, for example in the lungs of cystic fibrosis (CF) patients. Here, we discover that cell envelope protein homeostasis pathways underpin both antibiotic resistance and cross-protection in CF-associated bacteria. We find that inhibition of oxidative protein folding inactivates multiple species-specific resistance proteins. Using this strategy, we sensitize multidrug-resistant Pseudomonas aeruginosa to {beta}-lactam antibiotics and demonstrate promise of new treatment avenues for the recalcitrant emerging pathogen Stenotrophomonas maltophilia. The same approach also inhibits cross-protection between resistant S. maltophilia and susceptible P. aeruginosa, allowing eradication of both commonly co-occurring CF-associated organisms. Our results provide the basis for the development of next-generation strategies that target antibiotic resistance, while also impairing specific interbacterial interactions that enhance the severity of polymicrobial infections.

microbiology↗

bioRxiv2021

Breaking antimicrobial resistance by disrupting extracytoplasmic protein folding

Antimicrobial resistance in Gram-negative bacteria is one of the greatest threats to global health. New antibacterial strategies are urgently needed, and the development of antibiotic adjuvants that either neutralize resistance proteins or compromise the integrity of the cell envelope is of ever-growing interest. Most available adjuvants are only effective against specific resistance proteins. Here we demonstrate that disruption of cell envelope protein homeostasis simultaneously compromises several classes of resistance determinants. In particular, we find that impairing DsbA-mediated disulfide bond formation incapacitates diverse {beta}-lactamases and destabilizes mobile colistin resistance enzymes. Furthermore, we show that chemical inhibition of DsbA sensitizes multidrug-resistant clinical isolates to existing antibiotics and that the absence of DsbA, in combination with antibiotic treatment, substantially increases the survival of Galleria mellonella larvae infected with multidrug- resistant Pseudomonas aeruginosa. This work lays the foundation for the development of novel antibiotic adjuvants that function as broad-acting resistance breakers. IMPACT STATEMENTDisruption of disulfide bond formation sensitizes resistant Gram- negative bacteria expressing {beta}-lactamases and mobile colistin resistance enzymes to currently available antibiotics.

microbiology↗

bioRxiv2020

Novel structural components generate distinct type VI secretion system anchoring modes

The type VI secretion system (T6SS) is a phage-derived contractile nanomachine primarily involved in interbacterial competition. Its pivotal component, TssA, is indispensable for the assembly of the T6SS sheath structure, the contraction of which propels a payload of effector proteins into neighboring cells. Despite their key function, TssA proteins exhibit unexpected diversity and exist in two major forms, a short (TssAS) and a long (TssAL) TssA. Whilst TssAL proteins interact with a partner, called TagA, to anchor the distal end of the extended sheath, the mechanism for the stabilization of TssAS-containing T6SSs remains unknown. Here we discover a novel class of structural components that interact with short TssA proteins and contribute to T6SS assembly by stabilizing the polymerizing sheath from the baseplate. We demonstrate that the presence of these components is important for full sheath extension and optimal firing. Moreover, we show that the pairing of each form of TssA with a different class of sheath stabilization proteins results in T6SS apparatuses that either reside in the cell for a while or fire immediately after sheath extension, thus giving rise to different aggression behaviors. We propose that this functional diversity could contribute to the specialization of the T6SS to suit bacterial lifestyles in diverse environmental niches.

microbiology↗