RESUMEN
Biofilm formation is generally recognized as a bacterial defense mechanism against environmental threats, including antibiotics, bacteriophages, and leukocytes of the human immune system. Here, we show that for the human pathogen Vibrio cholerae, biofilm formation is not only a protective trait but also an aggressive trait to collectively predate different immune cells. We find that V. cholerae forms biofilms on the eukaryotic cell surface using an extracellular matrix comprising primarily mannose-sensitive hemagglutinin pili, toxin-coregulated pili, and the secreted colonization factor TcpF, which differs from the matrix composition of biofilms on other surfaces. These biofilms encase immune cells and establish a high local concentration of a secreted hemolysin to kill the immune cells before the biofilms disperse in a c-di-GMP-dependent manner. Together, these results uncover how bacteria employ biofilm formation as a multicellular strategy to invert the typical relationship between human immune cells as the hunters and bacteria as the hunted.
Asunto(s)
Vibrio cholerae , Animales , Humanos , Vibrio cholerae/metabolismo , Conducta Predatoria , Biopelículas , Fimbrias Bacterianas , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión GénicaRESUMEN
In the wild, bacteria are predominantly associated with surfaces as opposed to existing as free-swimming, isolated organisms. They are thus subject to surface-specific mechanics, including hydrodynamic forces, adhesive forces, the rheology of their surroundings, and transport rules that define their encounters with nutrients and signaling molecules. Here, we highlight the effects of mechanics on bacterial behaviors on surfaces at multiple length scales, from single bacteria to the development of multicellular bacterial communities such as biofilms.
Asunto(s)
Escherichia coli/fisiología , Pseudomonas aeruginosa/fisiología , Adhesión Bacteriana , Biopelículas , Transporte Biológico , Fenómenos Biomecánicos , Escherichia coli/citología , Locomoción , Pseudomonas aeruginosa/citologíaRESUMEN
Biofilms are a widely observed growth mode in which microbial communities are spatially structured and embedded in a polymeric extracellular matrix. Here, we focus on the model bacterium Vibrio cholerae and summarize the current understanding of biofilm formation, including initial attachment, matrix components, community dynamics, social interactions, molecular regulation, and dispersal. The regulatory network that orchestrates the decision to form and disperse from biofilms coordinates various environmental inputs. These cues are integrated by several transcription factors, regulatory RNAs, and second-messenger molecules, including bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). Through complex mechanisms, V. cholerae weighs the energetic cost of forming biofilms against the benefits of protection and social interaction that biofilms provide.
Asunto(s)
Biopelículas , Vibrio cholerae , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , GMP Cíclico/metabolismo , Regulación Bacteriana de la Expresión Génica , Factores de Transcripción/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/fisiologíaRESUMEN
Bacterial biofilms are among the most abundant multicellular structures on Earth and play essential roles in a wide range of ecological, medical, and industrial processes. However, general principles that govern the emergence of biofilm architecture across different species remain unknown. Here, we combine experiments, simulations, and statistical analysis to identify shared biophysical mechanisms that determine early biofilm architecture development at the single-cell level, for the species Vibrio cholerae, Escherichia coli, Salmonella enterica, and Pseudomonas aeruginosa grown as microcolonies in flow chambers. Our data-driven analysis reveals that despite the many molecular differences between these species, the biofilm architecture differences can be described by only 2 control parameters: cellular aspect ratio and cell density. Further experiments using single-species mutants for which the cell aspect ratio and the cell density are systematically varied, and mechanistic simulations show that tuning these 2 control parameters reproduces biofilm architectures of different species. Altogether, our results show that biofilm microcolony architecture is determined by mechanical cell-cell interactions, which are conserved across different species.
Asunto(s)
Biopelículas , Vibrio cholerae , Pseudomonas aeruginosa/genética , Vibrio cholerae/genética , Escherichia coli/genéticaRESUMEN
Fungi and bacteria often engage in complex interactions, such as the formation of multicellular biofilms within the human body. Knowledge about how interkingdom biofilms initiate and coalesce into higher-level communities and which functions the different species carry out during biofilm formation remain limited. We found native-state assemblages of Candida albicans (fungi) and Streptococcus mutans (bacteria) with highly structured arrangement in saliva from diseased patients with childhood tooth decay. Further analyses revealed that bacterial clusters are attached within a network of fungal yeasts, hyphae, and exopolysaccharides, which bind to surfaces as a preassembled cell group. The interkingdom assemblages exhibit emergent functions, including enhanced surface colonization and growth rate, stronger tolerance to antimicrobials, and improved shear resistance, compared to either species alone. Notably, we discovered that the interkingdom assemblages display a unique form of migratory spatial mobility that enables fast spreading of biofilms across surfaces and causes enhanced, more extensive tooth decay. Using mutants, selective inactivation of species, and selective matrix removal, we demonstrate that the enhanced stress resistance and surface mobility arise from the exopolymeric matrix and require the presence of both species in the assemblage. The mobility is directed by fungal filamentation as hyphae extend and contact the surface, lifting the assemblage with a "forward-leaping motion." Bacterial cell clusters can "hitchhike" on this mobile unit while continuously growing, to spread across the surface three-dimensionally and merge with other assemblages, promoting community expansion. Together, our results reveal an interkingdom assemblage in human saliva that behaves like a supraorganism, with disease-causing emergent functionalities that cannot be achieved without coassembly.
Asunto(s)
Biopelículas , Saliva , Streptococcus mutans , Candida albicans/metabolismo , Niño , Enfermedad , Humanos , Hifa/fisiología , Dinámica Poblacional , Saliva/microbiologíaRESUMEN
Bacteria often grow into matrix-encased three-dimensional (3D) biofilm communities, which can be imaged at cellular resolution using confocal microscopy. From these 3D images, measurements of single-cell properties with high spatiotemporal resolution are required to investigate cellular heterogeneity and dynamical processes inside biofilms. However, the required measurements rely on the automated segmentation of bacterial cells in 3D images, which is a technical challenge. To improve the accuracy of single-cell segmentation in 3D biofilms, we first evaluated recent classical and deep learning segmentation algorithms. We then extended StarDist, a state-of-the-art deep learning algorithm, by optimizing the post-processing for bacteria, which resulted in the most accurate segmentation results for biofilms among all investigated algorithms. To generate the large 3D training dataset required for deep learning, we developed an iterative process of automated segmentation followed by semi-manual correction, resulting in >18,000 annotated Vibrio cholerae cells in 3D images. We demonstrate that this large training dataset and the neural network with optimized post-processing yield accurate segmentation results for biofilms of different species and on biofilm images from different microscopes. Finally, we used the accurate single-cell segmentation results to track cell lineages in biofilms and to perform spatiotemporal measurements of single-cell growth rates during biofilm development.
Asunto(s)
Aprendizaje Profundo , Linaje de la Célula , Imagenología Tridimensional/métodos , Algoritmos , Biopelículas , Bacterias , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
Production of an extracellular matrix is essential for biofilm formation, as this matrix both secures and protects the cells it encases. Mechanisms underlying production and assembly of matrices are poorly understood. Vibrio cholerae, relies heavily on biofilm formation for survival, infectivity, and transmission. Biofilm formation requires Vibrio polysaccharide (VPS), which is produced by vps gene-products, yet the function of these products remains unknown. Here, we demonstrate that the vps gene-products vpsO and vpsU encode respectively for a tyrosine kinase and a cognate tyrosine phosphatase. Collectively, VpsO and VpsU act as a tyrosine phosphoregulatory system to modulate VPS production. We present structures of VpsU and the kinase domain of VpsO, and we report observed autocatalytic tyrosine phosphorylation of the VpsO C-terminal tail. The position and amount of tyrosine phosphorylation in the VpsO C-terminal tail represses VPS production and biofilm formation through a mechanism involving the modulation of VpsO oligomerization. We found that tyrosine phosphorylation enhances stability of VpsO. Regulation of VpsO phosphorylation by the phosphatase VpsU is vital for maintaining native VPS levels. This study provides new insights into the mechanism and regulation of VPS production and establishes general principles of biofilm matrix production and its inhibition.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Polisacáridos Bacterianos/biosíntesis , Multimerización de Proteína , Proteínas Tirosina Fosfatasas/metabolismo , Vibrio cholerae/fisiología , Proteínas Bacterianas/genética , Fosforilación/fisiología , Polisacáridos Bacterianos/genética , Proteínas Tirosina Fosfatasas/genéticaRESUMEN
Coordinated dynamics of individual components in active matter are an essential aspect of life on all scales. Establishing a comprehensive, causal connection between intracellular, intercellular, and macroscopic behaviors has remained a major challenge due to limitations in data acquisition and analysis techniques suitable for multiscale dynamics. Here, we combine a high-throughput adaptive microscopy approach with machine learning, to identify key biological and physical mechanisms that determine distinct microscopic and macroscopic collective behavior phases which develop as Bacillus subtilis swarms expand over five orders of magnitude in space. Our experiments, continuum modeling, and particle-based simulations reveal that macroscopic swarm expansion is primarily driven by cellular growth kinetics, whereas the microscopic swarming motility phases are dominated by physical cell-cell interactions. These results provide a unified understanding of bacterial multiscale behavioral complexity in swarms.
Asunto(s)
Bacillus subtilis/fisiología , Movimiento/fisiología , Comunicación Celular/fisiología , Proliferación Celular/fisiología , Cinética , Aprendizaje AutomáticoRESUMEN
Prokaryotic cells display a striking subcellular organization. Studies of the underlying mechanisms in different species have greatly enhanced our understanding of the morphological and physiological adaptation of bacteria to different environmental niches. The image analysis software tool BacStalk is designed to extract comprehensive quantitative information from the images of morphologically complex bacteria with stalks, flagella, or other appendages. The resulting data can be visualized in interactive demographs, kymographs, cell lineage plots, and scatter plots to enable fast and thorough data analysis and representation. Notably, BacStalk can generate demographs and kymographs that display fluorescence signals within the two-dimensional cellular outlines, to accurately represent their subcellular location. Beyond organisms with visible appendages, BacStalk is also suitable for established, non-stalked model organisms with common or uncommon cell shapes. BacStalk, therefore, contributes to the advancement of prokaryotic cell biology and physiology, as it widens the spectrum of easily accessible model organisms and enables highly intuitive and interactive data analysis and visualization.
Asunto(s)
Bacterias/metabolismo , Fenómenos Fisiológicos Bacterianos , Técnicas Citológicas/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Programas Informáticos , Biología Computacional/métodos , Análisis de Datos , Ensayos Analíticos de Alto Rendimiento/métodos , Quimografía/métodosRESUMEN
Vibrio cholerae is the causative agent of the diarrheal disease cholera, for which biofilm communities are considered to be environmental reservoirs. In endemic regions, and after algal blooms, which may result from phosphate enrichment following agricultural runoff, the bacterium is released from biofilms resulting in seasonal disease outbreaks. However, the molecular mechanism by which V. cholerae senses its environment and switches lifestyles from the biofilm-bound state to the planktonic state is largely unknown. Here, we report that the major biofilm scaffolding protein RbmA undergoes autocatalytic proteolysis via a phosphate-dependent induced proximity activation mechanism. Furthermore, we show that RbmA mutants that are defective in autoproteolysis cause V. cholerae biofilms to grow larger and mechanically stronger, correlating well with the observation that RbmA stability directly affects microbial community homeostasis and rheological properties. In conclusion, our biophysical study characterizes a novel phosphate-dependent breakdown pathway of RbmA, while microbiological data suggest a new, sensory role of this biofilm scaffolding element.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas/efectos de los fármacos , Compuestos de Magnesio/farmacología , Fosfatos/farmacología , Proteolisis , Vibrio cholerae/metabolismo , Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Vibrio cholerae/efectos de los fármacos , Vibrio cholerae/crecimiento & desarrolloRESUMEN
Bacterial biofilms are communities of bacteria that exist as aggregates that can adhere to surfaces or be free-standing. This complex, social mode of cellular organization is fundamental to the physiology of microbes and often exhibits surprising behavior. Bacterial biofilms are more than the sum of their parts: single-cell behavior has a complex relation to collective community behavior, in a manner perhaps cognate to the complex relation between atomic physics and condensed matter physics. Biofilm microbiology is a relatively young field by biology standards, but it has already attracted intense attention from physicists. Sometimes, this attention takes the form of seeing biofilms as inspiration for new physics. In this roadmap, we highlight the work of those who have taken the opposite strategy: we highlight the work of physicists and physical scientists who use physics to engage fundamental concepts in bacterial biofilm microbiology, including adhesion, sensing, motility, signaling, memory, energy flow, community formation and cooperativity. These contributions are juxtaposed with microbiologists who have made recent important discoveries on bacterial biofilms using state-of-the-art physical methods. The contributions to this roadmap exemplify how well physics and biology can be combined to achieve a new synthesis, rather than just a division of labor.
Asunto(s)
Adhesión Bacteriana/fisiología , Fenómenos Fisiológicos Bacterianos , Biopelículas , Percepción de Quorum/fisiología , Biopelículas/crecimiento & desarrolloRESUMEN
Recent advances in microscopy techniques make it possible to study the growth, dynamics, and response of complex biophysical systems at single-cell resolution, from bacterial communities to tissues and organoids. In contrast to ordered crystals, it is less obvious how one can reliably distinguish two amorphous yet structurally different cellular materials. Here, we introduce a topological earth mover's (TEM) distance between disordered structures that compares local graph neighborhoods of the microscopic cell-centroid networks. Leveraging structural information contained in the neighborhood motif distributions, the TEM metric allows an interpretable reconstruction of equilibrium and nonequilibrium phase spaces and embedded pathways from static system snapshots alone. Applied to cell-resolution imaging data, the framework recovers time ordering without prior knowledge about the underlying dynamics, revealing that fly wing development solves a topological optimal transport problem. Extending our topological analysis to bacterial swarms, we find a universal neighborhood size distribution consistent with a Tracy-Widom law.
Asunto(s)
Modelos Teóricos , Reconocimiento de Normas Patrones Automatizadas/métodos , Algoritmos , Animales , Fenómenos Biofísicos , Coloides/química , Microscopía por Crioelectrón , Drosophila , Entropía , Células Epiteliales/citología , Interpretación de Imagen Asistida por Computador/métodos , Modelos Biológicos , Modelos Químicos , ARN/químicaRESUMEN
Biofilms are a ubiquitous mode of microbial life and display an increased tolerance to different stresses. Inside biofilms, cells may experience both externally applied stresses and internal stresses that emerge as a result of growth in spatially structured communities. In this review, we discuss the spatial scales of different stresses in the context of biofilms, and if cells in biofilms respond to these stresses as a collection of individual cells, or if there are multicellular properties associated with the response. Understanding the organizational level of stress responses in microbial communities can help to clarify multicellular functions of biofilms.
Asunto(s)
Bacterias/metabolismo , Biopelículas , Bacterias/citología , Humanos , Estrés FisiológicoRESUMEN
Biofilms form when bacteria aggregate in a self-secreted exopolysaccharide matrix; they are resistant to antibiotics and implicated in disease. Nitric oxide (NO) is known to mediate biofilm formation in many bacteria via ligation to H-NOX (heme-NO/oxygen binding) domains. Most NO-responsive bacteria, however, lack H-NOX domain-containing proteins. We have identified another NO-sensing protein (NosP), which is predicted to be involved in two-component signaling and biofilm regulation in many species. Here, we demonstrate that NosP participates in the previously described H-NOX/NO-responsive multicomponent c-di-GMP signaling network in Shewanella oneidensis. Strains lacking either nosP or its co-cistronic kinase nahK (previously hnoS) produce immature biofilms, while hnoX and hnoK (kinase responsive to NO/H-NOX) mutants result in wild-type biofilm architecture. We demonstrate that NosP regulates the autophosphorylation activity of NahK as well as HnoK. HnoK and NahK have been shown to regulate three response regulators (HnoB, HnoC, and HnoD) that together comprise a NO-responsive multicomponent c-di-GMP signaling network. Here, we propose that NosP/NahK adds regulation on top of H-NOX/HnoK to modulate this c-di-GMP signaling network, and ultimately biofilm formation, by governing the flux of phosphate through both HnoK and NahK. In addition, it appears that NosP and H-NOX act to counter each other in a push-pull mechanism; NosP/NahK promotes biofilm formation through inhibition of H-NOX/HnoK signaling, which itself reduces the extent of biofilm formation. Addition of NO results in a reduction of c-di-GMP and biofilm formation, primarily through disinhibition of HnoK activity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas , GMP Cíclico/análogos & derivados , Óxido Nítrico/metabolismo , Shewanella/fisiología , Proteínas Bacterianas/genética , GMP Cíclico/metabolismo , Regulación Bacteriana de la Expresión Génica , Hemo/metabolismo , Shewanella/genética , Transducción de SeñalRESUMEN
Bacterial biofilms represent a major form of microbial life on Earth and serve as a model active nematic system, in which activity results from growth of the rod-shaped bacterial cells. In their natural environments, ranging from human organs to industrial pipelines, biofilms have evolved to grow robustly under significant fluid shear. Despite intense practical and theoretical interest, it is unclear how strong fluid flow alters the local and global architectures of biofilms. Here, we combine highly time-resolved single-cell live imaging with 3D multiscale modeling to investigate the mechanisms by which flow affects the dynamics of all individual cells in growing biofilms. Our experiments and cell-based simulations reveal three quantitatively different growth phases in strong external flow and the transitions between them. In the initial stages of biofilm development, flow induces a downstream gradient in cell orientation, causing asymmetrical dropletlike biofilm shapes. In the later developmental stages, when the majority of cells are sheltered from the flow by the surrounding extracellular matrix, buckling-induced cell verticalization in the biofilm core restores radially symmetric biofilm growth, in agreement with predictions of a 3D continuum model.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Modelos Biológicos , Vibrio cholerae/fisiología , MicrofluídicaRESUMEN
Biofilms are microbial collectives that occupy a diverse array of surfaces. It is well known that the function and evolution of biofilms are strongly influenced by the spatial arrangement of different strains and species within them, but how spatiotemporal distributions of different genotypes in biofilm populations originate is still underexplored. Here, we study the origins of biofilm genetic structure by combining model development, numerical simulations, and microfluidic experiments using the human pathogen Vibrio cholerae. Using spatial correlation functions to quantify the differences between emergent cell lineage segregation patterns, we find that strong adhesion often, but not always, maximizes the size of clonal cell clusters on flat surfaces. Counterintuitively, our model predicts that, under some conditions, investing in adhesion can reduce rather than increase clonal group size. Our results emphasize that a complex interaction between fluid flow and cell adhesiveness can underlie emergent patterns of biofilm genetic structure. This structure, in turn, has an outsize influence on how biofilm-dwelling populations function and evolve.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Modelos Biológicos , Adhesión Bacteriana/fisiología , Biología Computacional , Ingeniería Genética , Genotipo , Humanos , Hidrodinámica , Propiedades de Superficie , Vibrio cholerae/genética , Vibrio cholerae/patogenicidad , Vibrio cholerae/fisiologíaRESUMEN
Many bacterial species colonize surfaces and form dense 3D structures, known as biofilms, which are highly tolerant to antibiotics and constitute one of the major forms of bacterial biomass on Earth. Bacterial biofilms display remarkable changes during their development from initial attachment to maturity, yet the cellular architecture that gives rise to collective biofilm morphology during growth is largely unknown. Here, we use high-resolution optical microscopy to image all individual cells in Vibrio cholerae biofilms at different stages of development, including colonies that range in size from 2 to 4,500 cells. From these data, we extracted the precise 3D cellular arrangements, cell shapes, sizes, and global morphological features during biofilm growth on submerged glass substrates under flow. We discovered several critical transitions of the internal and external biofilm architectures that separate the major phases of V. cholerae biofilm growth. Optical imaging of biofilms with single-cell resolution provides a new window into biofilm formation that will prove invaluable to understanding the mechanics underlying biofilm development.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Análisis de la Célula Individual , Vibrio cholerae/fisiología , Vibrio cholerae/crecimiento & desarrolloRESUMEN
Biofilms are antibiotic-resistant, sessile bacterial communities that occupy most moist surfaces on Earth and cause chronic and medical device-associated infections. Despite their importance, basic information about biofilm dynamics in common ecological environments is lacking. Here, we demonstrate that flow through soil-like porous materials, industrial filters, and medical stents dramatically modifies the morphology of Pseudomonas aeruginosa biofilms to form 3D streamers, which, over time, bridge the spaces between obstacles and corners in nonuniform environments. We discovered that accumulation of surface-attached biofilm has little effect on flow through such environments, whereas biofilm streamers cause sudden and rapid clogging. We demonstrate that flow-induced shedding of extracellular matrix from surface-attached biofilms generates a sieve-like network that captures cells and other biomass, which add to the existing network, causing exponentially fast clogging independent of growth. These results suggest that biofilm streamers are ubiquitous in nature and strongly affect flow through porous materials in environmental, industrial, and medical systems.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Contaminación de Equipos , Pseudomonas aeruginosa/fisiología , Membranas Artificiales , PorosidadRESUMEN
Quorum sensing is a chemical communication process that bacteria use to regulate collective behaviors. Disabling quorum-sensing circuits with small molecules has been proposed as a potential strategy to prevent bacterial pathogenicity. The human pathogen Pseudomonas aeruginosa uses quorum sensing to control virulence and biofilm formation. Here, we analyze synthetic molecules for inhibition of the two P. aeruginosa quorum-sensing receptors, LasR and RhlR. Our most effective compound, meta-bromo-thiolactone (mBTL), inhibits both the production of the virulence factor pyocyanin and biofilm formation. mBTL also protects Caenorhabditis elegans and human lung epithelial cells from killing by P. aeruginosa. Both LasR and RhlR are partially inhibited by mBTL in vivo and in vitro; however, RhlR, not LasR, is the relevant in vivo target. More potent antagonists do not exhibit superior function in impeding virulence. Because LasR and RhlR reciprocally control crucial virulence factors, appropriately tuning rather than completely inhibiting their activities appears to hold the key to blocking pathogenesis in vivo.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Biopelículas/crecimiento & desarrollo , Pseudomonas aeruginosa/patogenicidad , Percepción de Quorum/fisiología , Transactivadores/antagonistas & inhibidores , Animales , Caenorhabditis elegans , Línea Celular , Escherichia coli , Humanos , Lactonas/química , Lactonas/farmacología , Análisis por Micromatrices , Estructura Molecular , Pseudomonas aeruginosa/fisiología , Piocianina , Percepción de Quorum/efectos de los fármacos , Mucosa Respiratoria/fisiología , Compuestos de Azufre/química , Compuestos de Azufre/farmacología , VirulenciaRESUMEN
Turbulence is ubiquitous, from oceanic currents to small-scale biological and quantum systems. Self-sustained turbulent motion in microbial suspensions presents an intriguing example of collective dynamical behavior among the simplest forms of life and is important for fluid mixing and molecular transport on the microscale. The mathematical characterization of turbulence phenomena in active nonequilibrium fluids proves even more difficult than for conventional liquids or gases. It is not known which features of turbulent phases in living matter are universal or system-specific or which generalizations of the Navier-Stokes equations are able to describe them adequately. Here, we combine experiments, particle simulations, and continuum theory to identify the statistical properties of self-sustained meso-scale turbulence in active systems. To study how dimensionality and boundary conditions affect collective bacterial dynamics, we measured energy spectra and structure functions in dense Bacillus subtilis suspensions in quasi-2D and 3D geometries. Our experimental results for the bacterial flow statistics agree well with predictions from a minimal model for self-propelled rods, suggesting that at high concentrations the collective motion of the bacteria is dominated by short-range interactions. To provide a basis for future theoretical studies, we propose a minimal continuum model for incompressible bacterial flow. A detailed numerical analysis of the 2D case shows that this theory can reproduce many of the experimentally observed features of self-sustained active turbulence.