generating-controlled-dynamic-chemical-landscapes-to-study-microbial

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

reconstruction-single-cell-innate-fluorescence-signatures-confocal

Reconstruction of Single-Cell Innate Fluorescence Signatures by Confocal Microscopy

Described here is confocal reflection microscopy-assisted single-cell innate fluorescence analysis (CRIF), a minimally invasive method for reconstructing ...

university of tsukuba

The way that gaseous metabolite production changes along with biofilm architecture development is poorly understood. To address this question, we developed a novel flow reactor biofilm culture method that allows for simultaneous assessment of gaseous metabolite production and architecture visualization. In this report, we establish the utility of this method using denitrification by Pseudomonas aeruginosa biofilms as a model system. Using this method, we were able to collect and analyze gaseous metabolites produced by denitrification and also visualize biofilm architecture in a nondestructive manner. Thus, we propose that this novel method is a powerful tool to investigate potential relationships between biofilm architecture and the gas-producing metabolic activity of biofilms, providing new insights into biofilm ecology.

Development of a novel biofilm continuous culture method for simultaneous assessment of architecture and gaseous metabolite production.

The feasibility of a method to nondestructively measure planktonic bacterial growth in a microfluidic device was addressed. Here, we report that the growth of Pseudomonas aeruginosa in a microfluidic device could be measured by a three-dimensional image analysis technique based on confocal reflection microscopy in a time-course.

Bacterial growth monitoring in a microfluidic device by confocal reflection microscopy.

The feasibility of a method to monitor biofilm development non-destructively in a microfluidic device was addressed. Here, we report that biofilm growth could be non-destructively monitored by an image analysis technique based on modification of confocal reflection microscopy.

Monitoring biofilm development in a microfluidic device using modified confocal reflection microscopy.

Continuous monitoring of ammonia removal by microbial complexes and observation of their morphology were carried out using a microdevice. Consumption of NH(4)(+) ions by active sludge could clearly be recorded over 48 h. Aggregation of the sludge could be observed in parallel by using confocal reflection microscopy.

Continuous monitoring of ammonia removal activity and observation of morphology of microbial complexes in a microdevice.

N-Octanoyl-L-homoserine lactone (C8-HSL) is an acyl-homoserine-lactone signal utilized in the quorum-sensing (QS) systems of Burkholderia cenocepacia and other bacterial species. Although also produced by Pseudomonas aeruginosa, its role in this species has not been elucidated. Here, we report that C8-HSL modulated antibiotic resistance and pyocyanin production in a P. aeruginosa efflux pump-deficient mutant. The rhl/las quorum-sensing system and qscR gene were both shown to be nonessential in the C8-HSL-induced changes in ofloxacin resistance, suggesting that P. aeruginosa possesses a distinct pathway to respond to C8-HSL.

The Response of Pseudomonas aeruginosa PAO1 Efflux Pump-Defective Mutants to N-Octanoyl-L-Homoserine Lactone.

Mass transport into the interior of biofilms and biological aggregates is a critical factor that affects their metabolic activity. In this study, we demonstrated the utility of a simple procedure that combines confocal reflection microscopy and fluorescent confocal laser scanning microscopy to visually explain the effects of biofilm structure on mass transport.

Visualizing the effects of biofilm structures on the influx of fluorescent material using combined confocal reflection and fluorescent microscopy.

A microfluidic device was developed for rapid determination of the minimum inhibitory concentration (MIC) of antibiotics against bacteria. A small volume of sample solution was introduced into multiple chambers simultaneously, and the growth of bacteria was quantified using a noninvasive three-dimensional (3D) visualization technique.

A microfluidic microbial culture device for rapid determination of the minimum inhibitory concentration of antibiotics.

Microbes interact with each other in multicellular communities and this interaction enables certain microorganisms to survive in various environments. Pseudomonas aeruginosa is a highly adaptable bacterium that ubiquitously inhabits diverse environments including soil, marine habitats, plants and animals. Behind this adaptivity, P. aeruginosa has abilities not only to outcompete others but also to communicate with each other to develop a multispecies community. In this review, we focus on how P. aeruginosa interacts with other microorganisms. P. aeruginosa secretes antimicrobial chemicals to compete and signal molecules to cooperate with other organisms. In other cases, it directly conveys antimicrobial enzymes to other bacteria using the Type VI secretion system (T6SS) or membrane vesicles (MVs). Quorum sensing is a central regulatory system used to exert their ability including antimicrobial effects and cooperation with other microbes. At least three quorum sensing systems are found in P. aeruginosa, Las, Rhl and Pseudomonas quinolone signal (PQS) systems. These quorum-sensing systems control the synthesis of extracellular antimicrobial chemicals as well as interaction with other organisms via T6SS or MVs. In addition, we explain the potential of microbial interaction analysis using several micro devices, which would bring fresh sensitivity to the study of interspecies interaction between P. aeruginosa and other organisms.

Interspecies interaction between Pseudomonas aeruginosa and other microorganisms.

Biofilms, such as dental plaque, are aggregates of microorganisms attached to a surface. Thus, visualization of biofilms together with their attached substrata is important in order to understand details of the interaction between them. However, so far there is limited availability of such techniques. Here, non-invasive visualization of biofilm formation with its attached substratum by applying the previously reported technique of continuous-optimizing confocal reflection microscopy (COCRM) is reported. The process of development of oral biofilm together with its substratum was sequentially visualized with COCRM. This study describes a convenient method for visualizing biofilm and its attached surface. 

Three-dimensional visualization of mixed species biofilm formation together with its substratum.

Biofilms are communities of surface-attached microbial cells that resist environmental stresses. In this study, we found that low concentrations of ethanol increase biofilm formation in Pseudomonas aeruginosa PAO1 but not in a mutant of it lacking both Psl and Pel exopolysaccharides. Low concentrations of ethanol also increased pellicle formation at the air-liquid interface. 

Low concentrations of ethanol stimulate biofilm and pellicle formation in Pseudomonas aeruginosa.

Cells respond to the environment and alter gene expression. Recent studies have revealed the social aspects of bacterial life, such as biofilm formation. Biofilm formation is largely affected by the environment, and the mechanisms by which the gene expression of individual cells affects biofilm development have attracted interest. Environmental factors determine the cell's decision to form or leave a biofilm. In addition, the biofilm structure largely depends on the environment, implying that biofilms are shaped to adapt to local conditions. Second messengers such as cAMP and c-di-GMP are key factors that link environmental factors with gene regulation. Cell-to-cell communication is also an important factor in shaping the biofilm. In this short review, we will introduce the basics of biofilm formation and further discuss environmental factors that shape biofilm formation. Finally, the state-of-the-art tools that allow us investigate biofilms under various conditions are discussed.

Environmental factors that shape biofilm formation.

Aquatic environments harbor a great diversity of microorganisms, which interact with the same patchy, particulate, or diffuse resources by means of a broad array of physiological and behavioral adaptations, resulting in substantially different life histories and ecological success. To date, efforts to uncover and understand this diversity have not been matched by equivalent efforts to identify unifying frameworks that can provide a degree of generality and thus serve as a stepping stone to scale up microscale dynamics to predict their ecosystem-level consequences. In particular, evaluating the ecological consequences of different resource landscapes and of different microbial adaptations has remained a major challenge in aquatic microbial ecology. Here, inspired by Ramon Margalef's mandala for phytoplankton, we propose a foraging mandala for microorganisms in aquatic environments, which accounts for both the local environment and individual adaptations. This biophysical framework distills resource acquisition into two fundamental parameters: the search time for a new resource and the growth return obtained from encounter with a resource. We illustrate the foraging mandala by considering a broad range of microbial adaptations and environmental characteristics. The broad applicability of the foraging mandala suggests that it could be a useful framework to compare disparate microbial strategies in aquatic environments and to reduce the vast complexity of microbe-environment interactions into a minimal number of fundamental parameters.

A Foraging Mandala for Aquatic Microorganisms.

Ephemeral aggregations of bacteria are ubiquitous in the environment, where they serve as hotbeds of metabolic activity, nutrient cycling, and horizontal gene transfer. In many cases, these regions of high bacterial concentration are thought to form when motile cells use chemotaxis to navigate to chemical hotspots. However, what governs the dynamics of bacterial aggregations is unclear. Here, we use an experimental platform to create realistic submillimeter-scale nutrient pulses with controlled nutrient concentrations. By combining experiments, mathematical theory, and agent-based simulations, we show that individual  bacteria begin chemotaxis toward hotspots of dissolved organic matter (DOM) when the magnitude of the chemical gradient rises sufficiently far above the sensory noise that is generated by stochastic encounters with chemoattractant molecules. Each DOM hotspot is surrounded by a dynamic ring of chemotaxing cells, which congregate in regions of high DOM concentration before dispersing as DOM diffuses and gradients become too noisy for cells to respond to. We demonstrate that  operates close to the theoretical limits on chemotactic precision. Numerical simulations of chemotactic bacteria, in which molecule counting noise is explicitly taken into account, point at a tradeoff between nutrient acquisition and the cost of chemotactic precision. More generally, our results illustrate how limits on sensory precision can be used to understand the location, spatial extent, and lifespan of bacterial behavioral responses in ecologically relevant environments.

Bacteria push the limits of chemotactic precision to navigate dynamic chemical gradients.

Here we analyzed the innate fluorescence signature of the single microbial cell, within both clonal and mixed populations of microorganisms. We found that even very similarly shaped cells differ noticeably in their autofluorescence features and that the innate fluorescence signatures change dynamically with growth phases. We demonstrated that machine learning models can be trained with a data set of single-cell innate fluorescence signatures to annotate cells according to their phenotypes and physiological status, for example, distinguishing a wild-type  cell from its nitrogen metabolism mutant counterpart and log-phase cells from stationary-phase cells of  We developed a minimally invasive method (onfocal eflection microscopy-assisted single-cell nnate luorescence [CRIF] analysis) to optically extract and catalog the innate cellular fluorescence signatures of each of the individual live microbial cells in a three-dimensional space. This technique represents a step forward from traditional techniques which analyze the innate fluorescence signatures at the population level and necessitate a clonal culture. Since the fluorescence signature is an innate property of a cell, our technique allows the prediction of the types or physiological status of intact and tag-free single cells, within a cell population distributed in a three-dimensional space. Our study presents a blueprint for a streamlined cell analysis where one can directly assess the potential phenotype of each single cell in a heterogenous population by its autofluorescence signature under a microscope, without cell tagging. A cell's innate fluorescence signature is an assemblage of fluorescence signals emitted by diverse biomolecules within a cell. It is known that the innate fluoresce signature reflects various cellular properties and physiological statuses; thus, they can serve as a rich source of information in cell characterization as well as cell identification. However, conventional techniques focus on the analysis of the innate fluorescence signatures at the population level but not at the single-cell level and thus necessitate a clonal culture. In the present study, we developed a technique to analyze the innate fluorescence signature of a single microbial cell. Using this novel method, we found that even very similarly shaped cells differ noticeably in their autofluorescence features, and the innate fluorescence signature changes dynamically with growth phases. We also demonstrated that the different cell types can be classified accurately within a mixed population under a microscope at the resolution of a single cell, depending solely on the innate fluorescence signature information. We suggest that single-cell autofluoresce signature analysis is a promising tool to directly assess the taxonomic or physiological heterogeneity within a microbial population, without cell tagging.

ArticleTotal : 2	Year
Publication title Cited by 4	2020
Publication title Cited by 1	2020

Article	Year
Development of a novel biofilm continuous culture method for simultaneous assessment of architecture and gaseous metabolite production. Applied and environmental microbiology\| PubMed ID: 18606794	2008
Bacterial growth monitoring in a microfluidic device by confocal reflection microscopy. Journal of bioscience and bioengineering\| PubMed ID: 20541131	2010
Monitoring biofilm development in a microfluidic device using modified confocal reflection microscopy. Journal of bioscience and bioengineering\| PubMed ID: 20547370	2010
Continuous monitoring of ammonia removal activity and observation of morphology of microbial complexes in a microdevice. Applied and environmental microbiology\| PubMed ID: 21515728	2011
The Response of Pseudomonas aeruginosa PAO1 Efflux Pump-Defective Mutants to N-Octanoyl-L-Homoserine Lactone. Microbes and environments\| PubMed ID: 21566394	2009
Visualizing the effects of biofilm structures on the influx of fluorescent material using combined confocal reflection and fluorescent microscopy. Microbes and environments\| PubMed ID: 21576853	2010
A microfluidic microbial culture device for rapid determination of the minimum inhibitory concentration of antibiotics. The Analyst\| PubMed ID: 23289096	2013
Interspecies interaction between Pseudomonas aeruginosa and other microorganisms. Microbes and environments\| PubMed ID: 23363620	2013
Three-dimensional visualization of mixed species biofilm formation together with its substratum. Microbiology and immunology\| PubMed ID: 23647374	2013
Low concentrations of ethanol stimulate biofilm and pellicle formation in Pseudomonas aeruginosa. Bioscience, biotechnology, and biochemistry\| PubMed ID: 25036502	2014
Environmental factors that shape biofilm formation. Bioscience, biotechnology, and biochemistry\| PubMed ID: 26103134	2016
A Foraging Mandala for Aquatic Microorganisms. The ISME journal\| PubMed ID: 30446738	2019
Bacteria push the limits of chemotactic precision to navigate dynamic chemical gradients. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 31097577	2019
Intra- and Interspecies Variability of Single-Cell Innate Fluorescence Signature of Microbial Cell. Applied and environmental microbiology\| PubMed ID: 31324624	2019

Yutaka Yawata

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