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The prediction of a stall precursor in an axial compressor is the basic guarantee to the stable operation of an aeroengine. How to predict and intelligently identify the instability of the system in advance is of great significance to the safety performance and active control of the aeroengine. In this paper, an aerodynamic system modeling method combination with the wavelet transform and gray wolf algorithm optimized support vector regression (WT-GWO-SVR) is proposed, which breaks through the fusion technology based on the feature correlation of chaotic data. Because of the chaotic characteristic represented by the sequence, the correlation-correlation (C-C) algorithm is adopted to reconstruct the phase space of the spatial modal. On the premise of finding out the local law of the dynamic system variety, the machine learning method is applied to model the reconstructed low-frequency components and high-frequency components, respectively. As the key part, the parameters of the SVR model are optimized by the gray wolf optimization algorithm (GWO) from the biological view inspired by the predatory behavior of gray wolves. In the definition of the hunting behaviors of gray wolves by mathematical equations, it is superior to algorithms such as differential evolution and particle swarm optimization. In order to further improve the prediction accuracy of the model, the multi-resolution and equivalent frequency distribution of the wavelet transform (WT) are used to train support vector regression. It is shown that the proposed WT-GWO-SVR hybrid model has a better prediction accuracy and reliability with the wavelet reconstruction coefficients as the inputs. In order to effectively identify the sign of the instability in the modeling system, a wavelet singular information entropy algorithm is proposed to detect the stall inception. By using the three sigma criteria as the identification strategy, the instability early warning can be given about 102r in advance, which is helpful for the active control.
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Bacteria can be genetically engineered to act as therapeutic delivery vehicles in the treatment of tumors, killing cancer cells or activating the immune system. This is known as bacteria-mediated cancer therapy (BMCT). Tumor invasion, colonization and tumor regression are major biological events, which are directly associated with antitumor effects and are uncontrollable due to the influence of tumor microenvironments during the BMCT process. Here, we developed a genetic circuit for dynamically programming bacterial lifestyles (planktonic, biofilm or lysis), to precisely manipulate the process of bacterial adhesion, colonization and drug release in the BMCT process, via hierarchical modulation of the lighting power density of near-infrared (NIR) light. The deep tissue penetration of NIR offers us a modality for spatio-temporal and non-invasive control of bacterial genetic circuits in vivo. By combining computational modeling with a high-throughput characterization device, we optimized the genetic circuits in engineered bacteria to program the process of bacterial lifestyle transitions by altering the illumination scheme of NIR. Our results showed that programming intratumoral bacterial lifestyle transitions allows precise control of multiple key steps throughout the BMCT process and therapeutic efficacy can be greatly improved by controlling the localization and dosage of therapeutic agents via optimizing the illumination scheme.
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Synthetic biology relies on the screening and quantification of genetic components to assemble sophisticated gene circuits with specific functions. Microscopy is a powerful tool for characterizing complex cellular phenotypes with increasing spatial and temporal resolution to library screening of genetic elements. Microscopy-based assays are powerful tools for characterizing cellular phenotypes with spatial and temporal resolution and can be applied to large-scale samples for library screening of genetic elements. However, strategies for high-throughput microscopy experiments remain limited. Here, we present a high-throughput, microscopy-based platform that can simultaneously complete the preparation of an 8 × 12-well agarose pad plate, allowing for the screening of 96 independent strains or experimental conditions in a single experiment. Using this platform, we screened a library of natural intrinsic promoters from Pseudomonas aeruginosa and identified a small subset of robust promoters that drives stable levels of gene expression under varying growth conditions. Additionally, the platform allowed for single-cell measurement of genetic elements over time, enabling the identification of complex and dynamic phenotypes to map genotype in high throughput. We expected that the platform could be employed to accelerate the identification and characterization of genetic elements in various biological systems, as well as to understand the relationship between cellular phenotypes and internal states, including genotypes and gene expression programs.
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The part of expression noise that is brought about by transcriptional regulation (represented here as NTR) is an important criterion for estimating the regulatory mode of a gene. However, characterization of NTR is an under-explored area, and there is little knowledge regarding the genome-wide NTR in the model pathogen Pseudomonas aeruginosa. Here, with a library of dual-color transcriptional reporters, we estimated the NTR for over 90% of the promoters in P. aeruginosa. Most promoters exhibit low NTR, while 42 and 115 promoters with high NTR were screened out in the exponential and the stationary growth phases, respectively. Specifically, a rearrangement of NTR was found in promoters involved in amino acid metabolism when bacteria enter the exponential phase. In addition, during the stationary phase, high NTR was found in a wide range of iron-related promoters involving siderophore synthesis and heme uptake, ExsA-regulated promoters involving bacterial virulence, and FleQ-regulated promoters involving biofilm development. We also found a large-scale negative dependence of transcriptional regulation between high-NTR promoters belonging to different functional categories. Our findings offer a global view of transcriptional heterogeneity in P. aeruginosa. IMPORTANCE The phenotypic diversity of Pseudomonas aeruginosa is frequently observed in research, suggesting that bacteria adopt strategies such as bet-hedging to survive ever-changing environments. Gene expression noise (GEN) is the major source of phenotypic diversity. Large GEN from transcriptional regulation (represented as NTR) represent an evolutionary necessity to maintain the copy number diversity of certain proteins in the population. Here, we provide a system-wide view of NTR in P. aeruginosa under nutrient-rich and stressed conditions. High NTR was found in genes involved in flagella biosynthesis and amino acid metabolism under both conditions. Specially, iron acquisition genes exhibited high NTR in the stressed condition, suggesting a great diversity of iron physiology in P. aeruginosa. We further revealed a global negative dependence of transcriptional regulation between those high-NTR genes under the stressed condition, suggesting a mutually exclusive relationship between different bacterial survival strategies.
Asunto(s)
Proteínas Bacterianas , Pseudomonas aeruginosa , Pseudomonas aeruginosa/genética , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica/genética , Hierro/metabolismo , Expresión Génica , Aminoácidos/genéticaRESUMEN
Single-cell behaviors are essential during early-stage biofilm formation. In this study, we aimed to evaluate whether single-cell behaviors could be precisely and continuously manipulated by optogenetics. We thus established adaptive tracking illumination (ATI), a novel illumination method to precisely manipulate the gene expression and bacterial behavior of Pseudomonas aeruginosa on the surface at the single-cell level by using the combination of a high-throughput bacterial tracking algorithm, optogenetic manipulation, and adaptive microscopy. ATI enables precise gene expression control by manipulating the optogenetic module gene expression and type IV pili (TFP)-mediated motility and microcolony formation during biofilm formation through bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) level modifications in single cells. Moreover, we showed that the spatial organization of single cells in mature biofilms could be controlled using ATI. Therefore, this novel method we established might markedly answer various questions or resolve problems in microbiology. KEY POINTS: ⢠High-resolution spatial and continuous optogenetic control of individual bacteria. ⢠Phenotype-specific optogenetic control of individual bacteria. ⢠Capacity to control biologically relevant processes in engineered single cells.
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Iluminación , Optogenética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas , GMP Cíclico/metabolismo , Fimbrias Bacterianas/genética , Guanosina Monofosfato/metabolismo , Optogenética/métodos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismoRESUMEN
The field of engineered living materials aims to construct functional materials with desirable properties of natural living systems. A recent study demonstrated the programmed self-assembly of bacterial populations by engineered adhesion. Here we use this strategy to engineer self-healing living materials with versatile functions. Bacteria displaying outer membrane-anchored nanobody-antigen pairs are cultured separately and, when mixed, adhere to each other to enable processing into functional materials, which we term living assembled material by bacterial adhesion (LAMBA). LAMBA is programmable and can be functionalized with extracellular moieties up to 545 amino acids. Notably, the adhesion between nanobody-antigen pairs in LAMBA leads to fast recovery under stretching or bending. By exploiting this feature, we fabricated wearable LAMBA sensors that can detect bioelectrical or biomechanical signals. Our work establishes a scalable approach to produce genetically editable and self-healable living functional materials that can be applied in biomanufacturing, bioremediation and soft bioelectronics assembly.
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Adhesión BacterianaRESUMEN
Bacterial pathogens operate by tightly controlling the pathogenicity to facilitate invasion and survival in host. While small molecule inducers can be designed to modulate pathogenicity to perform studies of pathogen-host interaction, these approaches, due to the diffusion property of chemicals, may have unintended, or pleiotropic effects that can impose limitations on their use. By contrast, light provides superior spatial and temporal resolution. Here, using optogenetics we reengineered GacS of the opportunistic pathogen Pseudomonas aeruginosa, signal transduction protein of the global regulatory Gac/Rsm cascade which is of central importance for the regulation of infection factors. The resultant protein (termed YGS24) displayed significant light-dependent activity of GacS kinases in Pseudomonas aeruginosa. When introduced in the Caenorhabditis elegans host systems, YGS24 stimulated the pathogenicity of the Pseudomonas aeruginosa strain PAO1 in a brain-heart infusion and of another strain, PA14, in slow killing media progressively upon blue-light exposure. This optogenetic system provides an accessible way to spatiotemporally control bacterial pathogenicity in defined hosts, even specific tissues, to develop new pathogenesis systems, which may in turn expedite development of innovative therapeutics.
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Proteínas Bacterianas/metabolismo , Caenorhabditis elegans/microbiología , Optogenética/métodos , Proteínas Quinasas/metabolismo , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidad , Transducción de Señal/genética , Factores de Transcripción/metabolismo , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/genética , Interacciones Huésped-Patógeno/genética , Luz , Microorganismos Modificados Genéticamente , Ingeniería de Proteínas/métodos , Proteínas Quinasas/genética , Pseudomonas aeruginosa/genética , Transducción de Señal/efectos de la radiación , Factores de Transcripción/genética , Virulencia/genética , Virulencia/efectos de la radiación , Factores de Virulencia/genéticaRESUMEN
Cyclic adenosine monophosphate (cAMP) is an important secondary messenger that controls carbon metabolism, type IVa pili biogenesis, and virulence in Pseudomonas aeruginosa. Precise manipulation of bacterial intracellular cAMP levels may enable tunable control of twitching motility or virulence, and optogenetic tools are attractive because they afford excellent spatiotemporal resolution and are easy to operate. Here, we developed an engineered P. aeruginosa strain (termed pactm) with light-dependent intracellular cAMP levels through introducing a photoactivated adenylate cyclase gene (bPAC) into bacteria. On blue light illumination, pactm displayed a 15-fold increase in the expression of the cAMP responsive promoter and an 8-fold increase in its twitching activity. The skin lesion area of nude mouse in a subcutaneous infection model after 2-day pactm inoculation was increased 14-fold by blue light, making pactm suitable for applications in controllable bacterial host infection. In addition, we achieved directional twitching motility of pactm colonies through localized light illumination, which will facilitate the studies of contact-dependent interactions between microbial species.
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Optogenética , Pseudomonas aeruginosa/metabolismo , Enfermedades Cutáneas Bacterianas/patología , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Luz , Ratones , Ratones Desnudos , Regiones Promotoras Genéticas , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/patogenicidad , Pseudomonas aeruginosa/efectos de la radiación , Enfermedades Cutáneas Bacterianas/microbiología , Virulencia/genéticaRESUMEN
Quantification of the rate of direct repeat deletion (DRD) is an important aspect in the research of DNA rearrangement. The widely used tetracycline selection method usually introduces antibiotic pressure to the tested organism, which may interfere with the DRD process. Also the length of repeat arm (LRA) is limited by the length of the TetR coding sequence. On the basis of the fluorescent microscopy and high-throughput imaging processing, here we developed a two-module genetic circuit, termed TFDEC (which stands for three-color fluorescence-based deletion event counter), to quantify the DRD rate under neutral conditions. DRD events were determined from the state of a three-state fluorescent logic gate constructed through coupling of an OR gate and an AND gate. TFDEC was applied in Pseudomonas aeruginosa, and we found that the DRD rate was RecA-dependent for long repeat arms (>500 bp) and RecA-independent for short repeat arms (<500 bp), which was consistent with the case in Escherichia coli. In addition, the increase of DRD rate followed an S-shaped curve with the increase of LRA, while treating cells with ciprofloxacin did not change the LRA-dependence of DRD. We also detected a significant increased DRD rate for long repeat arms in the uvrD (8-fold) and radA (4-fold) mutants. Our results show that the TFDEC method could be used as a complement tool for quantification of the DRD rate in the future.
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Pseudomonas aeruginosa/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Biología Sintética/métodos , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Mutación , Rec A Recombinasas/metabolismo , Eliminación de SecuenciaRESUMEN
Synthetic biology aims to make biology easier to engineer and focuses on the design and construction of core components that can be modeled, understood, and tuned to meet specific performance criteria, and the assembly of these smaller parts and devices into larger integrated systems to solve specific problems. Here, we designed and engineered a multicolor fluorescent reporter toolbox to simultaneously monitor the activities of multiple genes in single cells. The toolbox contained standardized and well-characterized genetic building blocks for the convenient and reproducible assembly of multiple promoter-reporter fusions (ranging from 1 to 4) into a single plasmid. Given the common problem of spectral crosstalk among multiple fluorescent proteins, we deciphered multiple spectral signatures within cells through a deduced linear unmixing algorithm. Our approach enabled the quantification of gene expression with direct FP concentrations, instead of mix-contributed fluorescence intensities, thus enabling true signal separation with high confidence. This approach performed well in the imaging of mixing cells with single FP labels. Additionally, combining with the multicolor toolbox, we succeeded in simultaneously monitoring the genetic dynamics of four selected quorum-sensing genes in response to the induction of two exogenously added autoinducers and were able to examine gene regulatory connections within the QS signaling network in Pseudomonas aeruginosa. Overall, this synthetic framework (i.e., the genetic toolbox and the well-evaluated approach of spectral correction) will be useful for applied synthetic biology projects, multicolor imaging, and analyzing interactions of multiple genes of natural genetic networks or assembling synthetic ones.
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Regulación Bacteriana de la Expresión Génica , Expresión Génica , Imagen Óptica/métodos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Percepción de Quorum/genética , Análisis de la Célula Individual/métodos , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Plásmidos/genética , Regiones Promotoras Genéticas , Biología Sintética/métodos , Proteína Fluorescente RojaRESUMEN
Pseudomonas aeruginosa can cause severe infections in humans. This bacterium often adopts a biofilm lifestyle that is hard to treat. In several previous studies, the PprA-PprB two-component system (TCS), which controls the expression of type IVb pili, BapA adhesin, and CupE fimbriae, was shown to be involved in biofilm formation (M. Romero, H. Silistre, L. Lovelock, V. J. Wright, K.-G. Chan, et al., Nucleic Acids Res 46:6823-6840, 2018, https://doi.org/10.1093/nar/gky324; S. de Bentzmann, C. Giraud, C. S. Bernard, V. Calderon, F. Ewald F, et al., PLoS Pathog 8:e1003052, 2012, https://doi.org/10.1371/journal.ppat.1003052). However, signals or environmental conditions that can trigger the PprA-PprB TCS are still unknown, and the molecular mechanisms of PprB-mediated biofilm formation are poorly characterized. Here, we report that carbon starvation stress (CSS) can induce the expression of pprB and genes in the PprB regulon. CSS-induced pprB transcription is mediated by the stress response sigma factor RpoS rather than the two-component sensor PprA. We also observed a strong negative regulation of PprB on the transcription of itself. Further experiments showed that PprB overexpression greatly enhanced cell-cell adhesion (CCA) and cell-surface adhesion (CSA) in P. aeruginosa Specifically, under the background of PprB overexpression, both the BapA adhesin and CupE fimbriae displayed positive effects on CCA and CSA, while the type IVb pili showed an unexpected negative effect on CCA and no effect on CSA. In addition, expression of the PprB regulon genes were significantly increased in 3-day colony biofilms, indicating a possible carbon limitation state. The CSS-RpoS-PprB-Bap/Flp/CupE pathway identified in this study provides a new perspective on the process of biofilm formation in carbon-limited environments.IMPORTANCE Typically, the determination of the external signals that can trigger a regulatory system is crucial to understand the regulatory logic and inward function of that system. The PprA-PprB two-component system was reported to be involved in biofilm formation in Pseudomonas aeruginosa, but the signals triggering this system are unknown. In this study, we found that carbon starvation stress (CSS) induces transcription of pprB and genes in the PprB regulon through an RpoS-dependent pathway. Increased PprB expression leads to enhanced cell-cell adhesion (CCA) and cell-surface adhesion (CSA) in P. aeruginosa Both CCA and CSA are largely dependent on the Bap secretion system and are moderately dependent on the CupE fimbriae. Our findings suggest that PprB reinforces the structure of biofilms under carbon-limited conditions, and the Bap secretion system and CupE fimbriae are two potential targets for biofilm treatment.
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Proteínas Bacterianas/genética , Biopelículas , Carbono/metabolismo , Genes Reguladores , Pseudomonas aeruginosa/genética , Adhesinas Bacterianas/genética , Fimbrias Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Factor sigmaRESUMEN
The second messenger signaling molecule cyclic diguanylate monophosphate (c-di-GMP) drives the transition between planktonic and biofilm growth in many bacterial species. Pseudomonas aeruginosa has two surface sensing systems that produce c-di-GMP in response to surface adherence. Current thinking in the field is that once cells attach to a surface, they uniformly respond by producing c-di-GMP. Here, we describe how the Wsp system generates heterogeneity in surface sensing, resulting in two physiologically distinct subpopulations of cells. One subpopulation has elevated c-di-GMP and produces biofilm matrix, serving as the founders of initial microcolonies. The other subpopulation has low c-di-GMP and engages in surface motility, allowing for exploration of the surface. We also show that this heterogeneity strongly correlates to surface behavior for descendent cells. Together, our results suggest that after surface attachment, P. aeruginosa engages in a division of labor that persists across generations, accelerating early biofilm formation and surface exploration.
Bacteria can adopt different lifestyles, depending on the environment in which they grow. They can exist as single cells that are free to explore their environment or group together to form 'biofilms'. The bacteria in biofilms stick to a surface, and produce a slimy 'matrix' that covers and thereby protects them. Biofilms have been found in lung infections that affect people with the genetic disorder cystic fibrosis, and can also form on the surface of medical implants. Because the biofilm lifestyle protects bacteria from the immune system and antimicrobial drugs, learning about how biofilms form could help researchers to discover ways to prevent and treat such infections. Many bacteria switch between the free-living and biofilm lifestyles by altering their levels of a signaling molecule called cyclic diguanylate monophosphate (called c-di-GMP for short). Bacteria living in biofilms have much higher levels of c-di-GMP than their free-living counterparts, and bacteria that have high levels of c-di-GMP produce more biofilm matrix. Bacteria called Pseudomonas aeruginosa use a protein signaling complex called the Wsp system to sense that they are on a surface and increase c-di-GMP production. Questions remained about how quickly this change in production occurs, and whether bacteria pass on their c-di-GMP levels to the new descendant cells when they divide. Armbruster et al. monitored individual cells of P. aeruginosa producing c-di-GMP as they began to form biofilms. Unexpectedly, not all cells increased their c-di-GMP levels when they first attached to a surface. Instead, Armbruster et al. found that there are two populations high and low c-di-GMP cells that each perform complementary and important tasks in the early stages of biofilm formation. The high c-di-GMP cells represent 'biofilm founders' that start to produce the biofilm matrix, whereas the low c-di-GMP cells represent 'surface explorers' that spend more time traveling along the surface. Armbruster et al. found that the Wsp surface sensing system generates these two populations of cells. Moreover, the c-di-GMP levels in a bacterial cell even affect the behavior of the descendant cells that form when it divides. This effect can persist for several cell generations. More work is needed to examine exactly how the biofilm founders and surface explorers interact and influence how biofilms form, and to discover if blocking c-di-GMP signaling prevents biofilm formation. This could ultimately lead to new strategies to prevent and treat infections in humans.
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Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , GMP Cíclico/análogos & derivados , Pseudomonas aeruginosa/metabolismo , Adhesión Bacteriana/genética , Adhesión Bacteriana/fisiología , Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , GMP Cíclico/metabolismo , Regulación Bacteriana de la Expresión Génica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiología , Percepción de Quorum/genéticaRESUMEN
The attachment of bacterial cells to a surface is implicated in the formation of biofilms. Although the surface-related behaviors in this process, such as single cell motility and surface sensing, have been investigated intensively, the precise information of separation distance between the attached cells and the surface has remained unclear. Here, we set a prism-based total internal reflection dark-field microscope (p-TIRDFM) combined with the microfluidic method to image the separation distance of single attached cells. We directly observed that bacterial cells attached to the surface with one nearest touchpoint, and it gradually changed to two touchpoints, respectively, for the two offspring with the cell division. We first monitored the fluctuation of the relative distance on nanometer scale when cells twitch on a surface and further established the relationship between the twitching velocity and the separation distance. The results indicated that the moving cells are a considerable distance apart from the surface and the separation distance fluctuated more widely than immobile cells.
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Pseudomonas aeruginosa/citología , Biopelículas , Técnicas Analíticas Microfluídicas , Microscopía Fluorescente , Imagen Óptica , Propiedades de SuperficieRESUMEN
We present a method capable of detecting single slow-growing and growth-arrested cells in a bacterial culture composed of physiologically and phenotypically different cells. Unlike the use of transcriptional reporters to gauge the metabolic activities in cells, here, we fuse two different fluorescent proteins with distinctive maturation rates to construct a timer to directly determine the growth rate of single Pseudomonas aeruginosa cells. We demonstrate that the dual-color fluorescent timer can indicate the slow-growing and growth-arrested cells from bacterial cultures in the presence of various environmental stresses, including nutrient starvation or antibiotic treatments, which greatly expand the methods for detecting and isolating persister cells.
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Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/crecimiento & desarrollo , Fluorescencia , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Cinética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismoRESUMEN
Exponentially growing bacteria in a well-mixed planktonic culture are generally assumed to be physiologically and phenotypically uniform and distinct from their genetically identical counterparts living in biofilms. Using a combination of high spatiotemporal microscopy and a bacterial tracking algorithm, in this study, we showed that planktonic cells of Pseudomonas aeruginosa differently attached to surfaces even when they remained in the exponential phase. We consistently observed that fast- and slow-attaching phenotypes coexist in planktonic cells, regardless of their growth phase. Furthermore, we found that (i) the distinct attaching phenotypes of planktonic cells resulted from the differential production of Psl and (ii) the RsmYZ/RsmA signaling pathway mainly regulated the differential production of Psl. Our results indicate that the differential production of Psl in P. aeruginosa plays a significant role in biofilm development and formation.IMPORTANCE The attachment of planktonic cells to surfaces is the first and most crucial step in biofilm formation. In this paper, we show that planktonic cells of Pseudomonas aeruginosa differently attach to surfaces. Typically, in the later exponential phase, approximately 80% of the cells can quickly attach to surfaces within 15 min, whereas approximately 20% of the cells slowly attach to surfaces, which greatly affects the initial stage of biofilm formation in the presence of flows. This is because fast-attaching cells are more likely to attach on surfaces to form microcolonies, whereas slow-attaching cells are more likely to remain in the mobile phase. This scenario is different from the previous understanding of biofilm formation in the initial stage, in which planktonic cells were thought to uniformly attach to surfaces. Most notably, the results of this study show that the different attachment manner of planktonic cells to surfaces affects the subsequent stages of biofilm formation. This research highlights that the phenotypic variations in planktonic cells plays significant roles in various stages of biofilm formation.
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Biopelículas/crecimiento & desarrollo , Fenotipo , Pseudomonas aeruginosa/metabolismo , Algoritmos , Adhesión Bacteriana/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Rastreo Celular , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Polisacáridos Bacterianos/genética , Polisacáridos Bacterianos/metabolismo , Pseudomonas aeruginosa/citología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crecimiento & desarrolloRESUMEN
Understanding the mechanisms that promote cooperative behaviors of bacteria in their hosts is of great significance to clinical therapies. Environmental stress is generally believed to increase competition and reduce cooperation in bacteria. Here, we show that bacterial cooperation can in fact be maintained because of environmental stress. We show that Pseudomonas aeruginosa regulates the secretion of iron-scavenging siderophores in the presence of different environmental stresses, reserving this public good for private use in protection against reactive oxygen species when under stress. We term this strategy "conditional privatization". Using a combination of experimental evolution and theoretical modeling, we demonstrate that in the presence of environmental stress the conditional privatization strategy is resistant to invasion by non-producing cheaters. These findings show how the regulation of public goods secretion under stress affects the evolutionary stability of cooperation in a pathogenic population, which may assist in the rational development of novel therapies.
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Antibiosis/fisiología , Hierro/metabolismo , Oligopéptidos/biosíntesis , Pseudomonas aeruginosa/fisiología , Sideróforos/biosíntesis , Simbiosis/fisiología , Antibacterianos/farmacología , Antibiosis/efectos de los fármacos , Antibiosis/efectos de la radiación , Evolución Biológica , Recuento de Colonia Microbiana , Fluoresceínas/química , Colorantes Fluorescentes/química , Oligopéptidos/metabolismo , Fotones/efectos adversos , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/efectos de la radiación , Sideróforos/metabolismo , Estrés Fisiológico , Simbiosis/efectos de los fármacos , Simbiosis/efectos de la radiación , Tobramicina/farmacologíaRESUMEN
In this paper, we present a new strategy for microprinting dense bacterial communities with a prescribed organization on a substrate. Unlike conventional bioprinting techniques that require bioinks, through optogenetic manipulation, we directly manipulated the behaviors of Pseudomonas aeruginosa to allow these living bacteria to autonomically form patterned biofilms following prescribed illumination. The results showed that through optogenetic manipulation, patterned bacterial communities with high spatial resolution (approximately 10 µm) could be constructed in 6 h. Thus, optogenetic manipulation greatly increases the range of available bioprinting techniques.
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Biopelículas , Bioimpresión/métodos , Optogenética/métodos , Pseudomonas aeruginosa/fisiología , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Luz , Microorganismos Modificados Genéticamente , Pseudomonas aeruginosa/genéticaRESUMEN
Synthetic biologists have attempted to solve real-world problems, such as those of bacterial biofilms, that are involved in the pathogenesis of many clinical infections and difficult to eliminate. To address this, we employed a blue light responding system and integrated it into the chromosomes of Pseudomonas aeruginosa. With making rational adaptions and improvements of the light-activated system, we provided a robust and convenient means to spatiotemporally control gene expression and manipulate biological processes with minimal perturbation in P. aeruginosa. It increased the light-induced gene expression up to 20-fold. Moreover, we deliberately introduced a functional protein gene PA2133 containing an EAL domain to degrade c-di-GMP into the modified system, and showed that the optimally engineered optogenetic tool inhibited the formation of P. aeruginosa biofilms through the induction of blue light, resulting in much sparser and thinner biofilms. Our approach establishes a methodology for leveraging the tools of synthetic biology to guide biofilm formation and engineer biofilm patterns with unprecedented spatiotemporal resolution. Furthermore, the findings suggest that the synthetic optogenetic system may provide a promising strategy that could be applied to control and fight biofilms.
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Biopelículas , Optogenética/métodos , Pseudomonas aeruginosa/fisiología , Proteínas Bacterianas/genética , Biopelículas/efectos de la radiación , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Histidina Quinasa/genética , Luz , Microscopía Fluorescente , Plásmidos/genética , Plásmidos/metabolismo , Imagen de Lapso de TiempoRESUMEN
Environmental bacteria persistently exist in hospitals and thereby often contaminate biomedical devices, which usually causes device-associated infections that have become a major cause of patient illness and death in the hospital. In this study, for the first time, the identification of strong shear flow persister (SSP) cells in Pseudomonas aeruginosa is reported. Unlike common persister cells that are highly tolerant to antibiotics, it is reported that the SSP cells can resist mechanical washings on the surfaces of various polymer materials and can form distinctive biofilms that are tolerant to high doses of aminoglycoside antibiotics. Most importantly, a general molecular mechanism is revealed by which an outer membrane protein crosslinks with polysaccharides to form gel-like adhesion complexes that can exert extremely strong adhesion strength (up to 50 N mm-2 ). Therefore, these findings are urgently required for ongoing research focused on preparing antifouling biomedical materials.