It is shown that deletion of mreB is viable in P. aeruginosa, but results in spherical cells that lose all forms of motility despite retaining flagella, and a previously overlooked polar effect of the in-frame mreB deletion on the downstream mreCD genes is uncovered.
Abstract
Despite growing interest in the MreBCD morphogenetic complex as a potential antimicrobial target, its function in Pseudomonas aeruginosa remains poorly understood. While previous studies using the MreB inhibitor A22 have established its role in cell shape maintenance and pilus regulation, the impact of mreB deletion has not been comprehensively investigated. Using genetic and microscopy-based approaches, we show that deletion of mreB is viable in P. aeruginosa, but results in spherical cells that lose all forms of motility despite retaining flagella. Importantly, we uncover a previously overlooked polar effect of the in-frame mreB deletion on the downstream mreCD genes and show, using CRISPRi-mediated silencing, that mreCD expression is essential for viability. ΔmreB mutants also display increased sensitivity to β-lactam antibiotics and enhanced initial surface attachment, yet form more compact biofilms with reduced dispersal. In mixed-culture biofilms, spherical ΔmreB cells are outcompeted by rod-shaped wild-type cells and remain confined to the biofilm base. The identification of natural P. aeruginosa isolates carrying truncated mreB alleles further indicates that loss of MreB function can be tolerated in natural populations. Together, our findings reveal important contributions of the MreBCD system to viability, morphogenesis, motility and biofilm development in P. aeruginosa, providing new insights into bacterial adaptation and informing the development of targeted antimicrobial strategies.
ABSTRACT Biofilm dispersion is a regulated process that enables bacteria to escape as free-living cells. This response coincides with matrix degradation, reduced cyclic di-GMP levels, heightened antibiotic susceptibility, and restored flagellar motility. While dispersed cells have been reported to be motile, the mechanisms enabling motility upon induction of dispersion remain unclear. Here, we explored the regulatory mechanism of how Pseudomonas aeruginosa biofilm cells switch their motility phenotype to disperse from biofilms. Our findings reveal that changes in motility gene expression are initiated at the periphery of biofilms during induced dispersion, in which increased expression of the flagellar gene fliC is controlled by the phosphodiesterase (PDE) DipA and the chemosensory protein BdlA. In response to the dispersion signal, BdlA forms dynamic clusters and relocates to the flagellated cell pole, where it interacts with and stimulates the PDE activity of DipA, ultimately leading to elevated fliC expression. These findings establish a mechanistic link between signal sensing and motility reversion, demonstrating that the spatiotemporal coordination of BdlA and DipA governs the motility switch during P. aeruginosa biofilm dispersion. IMPORTANCE Our findings reveal a new regulatory mechanism in which dispersion signals drive BdlA relocation, activate DipA’s phosphodiesterase activity, lower cyclic di-GMP, and elevate fliC expression. Moreover, by showing that BdlA foci formation and co-localization with DipA occur in response to dispersion signals, we demonstrate that the spatiotemporal regulation of BdlA and DipA extends the Touch-Seed-and-Go model beyond surface attachment to encompass dispersion and, thus, the reversion from a sessile to a motile growth state. Our findings reveal a new regulatory mechanism in which dispersion signals drive BdlA relocation, activate DipA’s phosphodiesterase activity, lower cyclic di-GMP, and elevate fliC expression. Moreover, by showing that BdlA foci formation and co-localization with DipA occur in response to dispersion signals, we demonstrate that the spatiotemporal regulation of BdlA and DipA extends the Touch-Seed-and-Go model beyond surface attachment to encompass dispersion and, thus, the reversion from a sessile to a motile growth state.
Soyoung Park, Karin Sauer· Journal of Bacteriology· 0 citations
Hypervirulent Klebsiella pneumoniae (hvKp) is an emerging threat due to its capacity to cause severe community-acquired infections and its increasing convergence with multidrug resistance. However, functional assessment of hvKp virulence still relies largely on mammalian infection models, which are costly, ethically constrained, and poorly suited for scalable early-stage screening. Here, we establish a proof-of-concept cell-based biosensing framework using the social amoeba Dictyostelium discoideum to detect capsule-associated virulence phenotypes in the model hvKp strain SGH10. Using SGH10 and a scarless capsule-null ΔwcaJ mutant, we show that loss of capsule production strongly reduces mucoviscosity, zebrafish lethality, resistance to amoebal predation, inhibition of social development, and impairment of amoebal motility. These host behavioral responses were quantified through complementary readouts, including predation plaque expansion, multicellular development, fluorescence microscopy, live-cell tracking, and exploratory multivariate integration. Compared with SGH10, the ΔwcaJ mutant produced phenotypes resembling those of the avirulent control strain KpGe, supporting capsule production as a major driver of phagocytosis resistance and host-response disruption in this model. Rather than providing a broadly validated classifier for hvKp, our results define a calibrated experimental framework in which D. discoideum functions as a living phenotypic sensor for capsule-dependent virulence traits. This platform offers a low-cost, genetically tractable, and imaging-compatible system for dissecting hvKp host interactions and for guiding future validation of scalable virulence biosensing across larger and genetically diverse K. pneumoniae collections.
Ian Pérez-Ramírez, Matías Gálvez-Silva, Diego Rojas et al.· ACS Sensors· 0 citations
ABSTRACT Bacteria employ antagonistic strategies to eliminate competitors of an ecological niche. Contact-dependent mechanisms, such as the type VI secretion system (T6SS), are prevalent in host-associated bacteria, yet we know relatively little about how T6SS+ strains make contact with competitors in highly viscous environments, such as host mucus. To better understand how cells respond to and contact one another in such environments, we performed a genome-wide transposon mutant screen of the T6SS-wielding beneficial bacterial symbiont, Vibrio fischeri MJ11, and identified two sets of genes that are conditionally required for killing. We found that surface modification and flagellar-associated genes do not affect T6SS directly and are therefore not required for interbacterial killing when cell contact is forced, yet are necessary for killing in high-viscosity liquid (hydrogel), where cell-cell contact must be biologically mediated. Quantitative transcriptomics revealed that V. fischeri significantly increases expression of both T6SS genes and cell surface modification factors upon transition from low- to high-viscosity media. Consistent with coincubation and fluorescence microscopy data, flagella are not required for T6SS expression in hydrogel. However, flagella were necessary to enhance expression of ~50% of the genome in hydrogel, including the surface modification genes identified in our screen and functional pathways important for host colonization, such as uptake of host-relevant iron and carbon sources, and nitric oxide detoxification enzymes. Our findings suggest that flagella play a key role when V. fischeri cells coordinately activate competitive strategies and host colonization factors, underscoring the significance of the physical environment in directing complex bacterial behaviors. IMPORTANCE The physical environment has dramatic effects on bacterial behavior, but little is known about how mechanical signals impact antagonistic interactions. Symbiotic bacteria use molecular weapons to eliminate competitors for limited space within highly viscous host tissue and mucus. Previously, we found that a putative lipoprotein adhesin, TasL, and an unknown ligand are required to bring competitor cells within range of the T6SS weapon. Here, we found that mutations in flagella or predicted surface modification genes prevent TasL-mediated adhesion and killing in high viscosity. Transcriptomics revealed the flagella are required to coordinate expression of host colonization factors with the T6SS interbacterial weapon when transitioning from lower to higher viscosity conditions. These findings suggest that flagella may play a role in sensing mechanical signals, such as environmental viscosity, to enhance a symbiont’s ability to successfully colonize the host while efficiently eliminating potential competitors from the host niche. The physical environment has dramatic effects on bacterial behavior, but little is known about how mechanical signals impact antagonistic interactions. Symbiotic bacteria use molecular weapons to eliminate competitors for limited space within highly viscous host tissue and mucus. Previously, we found that a putative lipoprotein adhesin, TasL, and an unknown ligand are required to bring competitor cells within range of the T6SS weapon. Here, we found that mutations in flagella or predicted surface modification genes prevent TasL-mediated adhesion and killing in high viscosity. Transcriptomics revealed the flagella are required to coordinate expression of host colonization factors with the T6SS interbacterial weapon when transitioning from lower to higher viscosity conditions. These findings suggest that flagella may play a role in sensing mechanical signals, such as environmental viscosity, to enhance a symbiont’s ability to successfully colonize the host while efficiently eliminating potential competitors from the host niche.
Lauren Speare, Liang Zhao, Morgan N. Pavelsky et al.· mBio· 0 citations
ABSTRACT Evolutionary constraints governing flagellar number in bacterial pathogens remain poorly understood. While related Pseudomonas species are hyperflagellated, Pseudomonas aeruginosa maintains strict monoflagellation through the FleQ-FleN regulatory circuit. Here, we demonstrate that FleN dosage is essential for maintaining monoflagellation and fitness. Confirming earlier reports, wild-type P. aeruginosa displayed unipolar monoflagellation, whereas ∆fleN mutants developed multiple flagella, frequently more than four, in unipolar or bipolar arrangements. These hyperflagellated cells showed marked fitness defects, including reduced growth, attenuated virulence in a nematode infection model, and a competitive disadvantage in co-culture. The virulence defect reflected hyperflagellation rather than the loss of motility, since a non-flagellated ∆fliC mutant retained full pathogenicity. Notably, ∆fleN cells rapidly evolved suppressor mutations in fleQ that partially restored growth and motility without always restoring monoflagellation. Five independent suppressor alleles mapped to key FleQ domains, four in the AAA+ ATPase domain, and one in the DNA-binding domain, consistent with reduced FleQ activity that rebalances the circuit. Single-cell holographic tracking showed that suppressor strains swim with heterogeneous dynamics, including subpopulations that reach wild-type (WT) speeds, despite carrying multiple flagella. Quantitative proteomics indicated that the fitness burden extends beyond flagellar components, with protein-abundance changes across metabolism, stress responses, and signaling. Conversely, selection under high-viscosity conditions drove wild-type cells to acquire enhanced spreading through a recurrent fleN mutation, fleNV178G; multiflagellation is therefore accessible. Together, these findings indicate that the FleQ-FleN circuit balances the flagellar number against fitness, holding P. aeruginosa at a single flagellum while retaining the latent capacity to reach multiflagellated states through single mutations. IMPORTANCE Bacterial flagella are extracellular appendages that rotate to propel the cell and enable swimming motility. While some bacteria have multiple flagella, many pathogenic species like Pseudomonas aeruginosa have just one. Surprisingly, mutants of P. aeruginosa with multiple flagella performed worse, that is, they grew more slowly, were less infectious in laboratory animals, and were outcompeted by wild-type bacteria. Even when some mutant bacteria evolved compensatory changes, they still struggled compared to single-flagellum bacteria. This reveals an important evolutionary trade-off: while multiple flagella might seem advantageous for movement, having just one flagellum allows the bacteria to grow faster and cause more severe infections. This plasticity likely explains why P. aeruginosa is so successful both in the environment and as a human pathogen. Bacterial flagella are extracellular appendages that rotate to propel the cell and enable swimming motility. While some bacteria have multiple flagella, many pathogenic species like Pseudomonas aeruginosa have just one. Surprisingly, mutants of P. aeruginosa with multiple flagella performed worse, that is, they grew more slowly, were less infectious in laboratory animals, and were outcompeted by wild-type bacteria. Even when some mutant bacteria evolved compensatory changes, they still struggled compared to single-flagellum bacteria. This reveals an important evolutionary trade-off: while multiple flagella might seem advantageous for movement, having just one flagellum allows the bacteria to grow faster and cause more severe infections. This plasticity likely explains why P. aeruginosa is so successful both in the environment and as a human pathogen.
Analí Migueles-Lozano, Merrill Asp, Sofia T Rocha et al.· mBio· 0 citations
Pseudomonas aeruginosa is an opportunistic pathogen in which motility, biofilm formation, and stress adaptation contribute to virulence. Drug repurposing represents a practical strategy for identifying compounds that influence these processes. In this study, the effects of 5-fluorouracil (5-FU) on motility, extracellular DNA (eDNA) production, and gene expression were examined in P. aeruginosa PAO1. Swimming, swarming, and twitching motility assays were performed in the presence of increasing concentrations of 5-FU. Transcriptional responses of motility, rhamnolipid, and DNA repair-associated genes were evaluated using quantitative real-time PCR. eDNA levels were quantified using fluorescence-based assays and visualized by confocal laser scanning microscopy. Exposure to 5-FU resulted in concentration-dependent reductions in swimming, swarming, and twitching motility. These changes were associated with downregulation of multiple genes involved in flagellar and type IV pili function, including motA, flhA, fliD, pilA, and pilI, and reduced expression of lasB and rhlAB. At 24 h, eDNA levels were decreased relative to untreated controls, whereas at 48 h, higher concentrations of 5-FU were associated with increased eDNA accumulation and upregulation of several DNA repair genes, including xthA, nth, recJ, sbcB, and eddB. These results indicate the multifaceted effects of 5-FU on important virulence factors of P. aeruginosa.
Amani A Niazy, May M. Alrashed, R. Lambarte et al.· PLoS ONE· 0 citations
Xenogeneic silencers, such as histone-like nucleoid structuring protein (H-NS), are critical for maintaining horizontally acquired genes in bacterial genomes and minimizing fitness costs associated with inappropriate expression. For bacterial pathogens, this has enabled the acquisition of costly virulence regulons, with H-NS balancing the need for tight silencing with rapid expression in host environments. For Salmonella enterica serovar Typhimurium (STm), survival in these environments relies on phenotypic heterogeneity in virulence gene expression and evolutionary adaptation. Although complete loss of hns is highly deleterious in STm, how subtle impairments to this global silencer disrupt heterogeneity in virulence gene expression and alter adaptation to host environments remains poorly understood. Here, we identify an STm hns hypomorph strain and find that its reduced H-NS DNA-binding affinity increases the proportion of virulence-expressing cells, resulting in enhanced epithelial cell infection in vitro. Furthermore, through experimental evolution in intracellular-like conditions in vitro, we demonstrate that both wild-type and mutant populations converge on disrupting the SPI-2 virulence regulon to improve fitness; however, the mutant population also acquires distinct adaptive mutations to resolve the underlying dysregulation in gene expression. These results suggest that H-NS sets single-cell virulence activation thresholds and that even minor disruptions to its silencing function impact pathogen adaptation, highlighting its role as a critical evolutionary buffer.
Leah Mclelland, Madison Spratt, Dakshya Karki et al.· PLoS Pathogens· 0 citations