A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.Download video file.^{(97M, mp4)}     

Clonal Growth Strategies Along Flooding and Grazing Gradients in Atlantic Coastal Meadows

Specific composition and species clonal traits were characterized along combined flooding and grazing gradients to answer two questions. i) To what extent does the interaction of flooding and grazing influence the clonal characteristics of the vegetation? ii) Are the effects of both environmental factors independent or interactive? This study was carried out in a wet meadow along the Atlantic coast (France). Three plant communities (hygrophilous, meso-hygrophilous and mesophilous) were distinguished along a flooding gradient and five levels of grazing pressure were controlled through an experimental design (from no grazing to heavy grazing). We monitored species composition and retrieved, for each species, the type of clonal growth organs (CGOs) and clonal traits from the CLO-PLA3 database. We identified two syndromes of clonal traits: ??above-ground splitters?? and ??below-ground integrators??. Clonal traits played a key role in plant assembly in the studied meadows. The interaction of both environmental factors selected for particular syndromes of clonal traits; however, flooding had a stronger filtering effect than grazing. The hygrophilous community was dominated by above-ground splitters, whereas the meso-hygrophilous vegetation was dominated by below-ground integrators. In the mesophilous community, clonal composition was the most diverse and shared clonal traits with the vegetation of both the hygrophilous and meso-hygrophilous communities. Grazing impact on CGOs and clonal traits differed between plant communities, i.e., the effect of grazing was modulated by the flooding regime. This study confirmed that vegetation responses to grazing might depend on the pool of traits, primarily filtered by environmental factors such as flooding.     

Adaptive-Control Model for Neutrophil Orientation in the Direction of Chemical Gradients

Neutrophils have a remarkable ability to detect the direction of chemoattractant gradients and move directionally in response to bacterial infections and tissue injuries. For their role in health and disease, neutrophils have been extensively studied, and many of the molecules involved in the signaling mechanisms of gradient detection and chemotaxis have been identified. However, the cellular-scale mechanisms of gradient sensing and directional neutrophil migration have been more elusive, and existent models provide only limited insight into these processes. Here, we propose a what we believe is a novel adaptive-control model for the initiation of cell polarization in response to gradients. In this model, the neutrophils first sample the environment by extending protrusions in random directions and subsequently adapt their sensitivity depending on localized, temporal changes in stimulation levels. Our results suggest that microtubules may play a critical role in integrating all the sensing events from the cellular periphery through their redistribution inside the neutrophils, and may also be involved in modulating local signaling. An unexpected finding was that model neutrophils exhibit significant randomness in timing and directionality of activation, comparable to our experimental observations in microfluidic devices. Moreover, their responses are robust against alterations of the rate and amplitude of the signaling reactions, and for a broad range in chemoattractant concentrations and spatial gradients.     

Flow-Based Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable,Competing Gradients

Chemotaxis is the migration of cells in gradients of chemoeffector molecules. Although multiple, competing gradients must often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to gradients of multiple signals. In this work, we developed a microfluidic device for generating precise and stable gradients of signaling molecules. We used the device to investigate the effects of individual and combined chemoeffector gradients on Escherichia coli chemotaxis. Laminar flow-based diffusive mixing was used to generate gradients, and the chemotactic responses of cells expressing green fluorescent protein were determined using fluorescence microscopy. Quantification of the migration profiles indicated that E. coli was attracted to the quorum-sensing molecule autoinducer-2 (AI-2) but was repelled from the stationary-phase signal indole. Cells also migrated toward higher concentrations of isatin (indole-2,3-dione), an oxidized derivative of indole. Attraction to AI-2 overcame repulsion by indole in equal, competing gradients. Our data suggest that concentration-dependent interactions between attractant and repellent signals may be important determinants of bacterial colonization of the gut.Bacteria sense chemoeffectors using cell surface receptors (13, 29). Cells constantly monitor the concentration of specific molecules, comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement (6, 22). Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Natural habitats for Escherichia coli, such as the gastrointestinal (GI) tract, are typically heterogeneous and contain multiple chemoeffectors with potentially opposing effects. The integrated chemotactic response in such environments is thus likely to be an important factor in bacterial colonization.Conventional approaches for investigating bacterial chemotaxis, such as the swim plate and capillary (1) assays, are not ideal for quantifying bacterial migration. Chemotactic-ring formation in semisolid agar requires metabolizable attractants and is subject to multiple factors, and both it and the traditional capillary assay are poorly designed to investigate repellent taxis. Mao et al. (23) were the first to investigate bacterial taxis in a microfluidic flow cell. In their device, a concentration gradient is formed by the diffusive mixing of two inlet streams. However, the exposure to a fully developed gradient in this device is limited because it takes time for the gradient to develop.Variations of this technique, such as three-channel microfluidic devices (7, 8) in which a linear gradient is generated in the absence of flow or a T-channel device that monitors chemotaxis perpendicular to the direction of fluid flow (18), were developed subsequently. The T-channel system has many of the limitations of the device developed by Mao et al. (23), and nonflow systems, like the capillary assay (1), suffer from a lack of temporal stability of the gradients.Here, we report a flow-based microfluidic chemotaxis device that is coupled to a gradient generator. Bacteria are exposed to precise and temporally stable concentration gradients of chemoeffectors over the length of the microfluidic channel. This device was used to quantify E. coli chemotaxis in response to the canonical chemoeffectors l-aspartate and Ni²⁺. The device was also used to investigate chemotaxis toward cell-cell communication signals such as autoinducer-2 (AI-2), indole, and isatin that are likely to be present in the in vivo microenvironment in which E. coli is present (e.g., the human GI tract). The data obtained reinforce the idea that concentration-dependent interactions between different chemical signals could be important determinants of bacterial colonization in natural environments.     

宠物重要人兽共患细菌病流行现状及其控制策略

随着我国社会经济发展和城市化进程的加速,宠物数量日益增多,宠物人兽共患病发病率日渐增高,本文对我国宠物重要人兽共患细菌病流行状况、原因进行了阐述,并提出了防控对策。     

Tracking Shallow Chemical Gradients by Actin-Driven Wandering of the Polarization Site

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Highlights? A wandering polarity site distributes growth in yeast cells responding to pheromone ? Wandering, but not polarity establishment, is actin dependent ? A mathematical model incorporating polarity and vesicle traffic exhibits wandering ? Blocking wandering impairs gradient tracking     

Bacterial Community Structure Along Moisture Gradients in the Parafluvial Sediments of Two Ephemeral Desert Streams

Microorganisms inhabiting stream sediments mediate biogeochemical processes of importance to both aquatic and terrestrial ecosystems. In deserts, the lateral margins of ephemeral stream channels (parafluvial sediments) are dried and rewetted, creating periodically wet conditions that typically enhance microbial activity. However, the influence of water content on microbial community composition and diversity in desert stream sediments is unclear. We sampled stream margins along gradients of wet to dry sediments, measuring geochemistry and bacterial 16S rRNA gene composition, at streams in both a cold (McMurdo Dry Valleys, Antarctica) and hot (Chihuahuan Desert, New Mexico, USA) desert. Across the gradients, sediment water content spanned a wide range (1.6–37.9% w/w), and conductivity was highly variable (12.3–1,380 μS cm⁻²). Bacterial diversity (at 97% sequence similarity) was high and variable, but did not differ significantly between the hot and cold desert and was not correlated with sediment water content. Instead, conductivity was most strongly related to diversity. Water content was strongly related to bacterial 16S rRNA gene community composition, though samples were distributed in wet and dry clusters rather than as assemblages shifting along a gradient. Phylogenetic analyses showed that many taxa from wet sediments at the hot and cold desert site were related to, respectively, halotolerant Gammaproteobacteria, and one family within the Sphingobacteriales (Bacteroidetes), while dry sediments at both sites contained a high proportion of taxa related to the Acidobacteria. These results suggest that bacterial diversity and composition in desert stream sediments is more strongly affected by hydrology and conductivity than temperature.     

细菌病原体突破天然免疫系统的策略

近10年来,天然免疫和病原微生物两个相关领域研究取得了巨大进展。面对功能完备的天然免疫系统,一些病原微生物感染宿主的机制逐步进化了,并且病原体通过此进化避免被识别,促进或避免炎症的产生。事实证明,病原体不仅能在天然免疫反应中幸存,而且还能利用天然免疫反应来提高它的致病性。     

Wave Forms of Caenorhabditis elegans in a Chemical Attractant and Repellent and in Thermal Gradients

The wave forms and activity patterns of Caenorhabditis elegans were examined on agar in the presence of known chemical attractants (NaCl) and repellents (D-tryptophan), and in thermal gradients. Total activity was reduced in both attractants and repellents. Different combinations of transfers between chemicals were investigated. Two thresholds were found for NaCl: 10^-3 M NaC1 caused reduced activity; 10⁻⁵ M NaCl increased reversals. D- or L-tryptophan influenced neither orientation nor the ability of thermally acclimatized individuals to remain at their eccritic temperature.     

The Impact of Bacterial Diet on the Migration and Navigation of Caenorhabditis elegans

Can diet have a significant impact on the ability of organisms to sense and locate food? Focusing on the bacterial feeder Caenorhabditis elegans, we investigated what effect preconditioning on a range of bacterial substrates had on the subsequent chemotaxis process involved in the nematode locating other bacterial populations. Remarkably, we found that C. elegans, initially fed on a diet of Escherichia coli OP50, was significantly impaired in finding E. coli OP50 populations, compared to other available bacterial populations (P < 0.001). We found similar results for another bacterial feeding nematode species, suggesting that a general “substrate legacy” may operate across a wide range of organisms. We discuss this important finding with respect to the variation in response exhibited within a given nematode population, and the impact nematode migration has on bacterial dispersal in the environment.     

Composite Bacterial Hopanoids and Their Microbial Producers across Oxygen Gradients in the Water Column of the California Current

Hopanoids are pentacyclic triterpenoid lipids produced by many prokaryotes as cell membrane components. The structural variations of composite hopanoids, or bacteriohopanepolyols (BHPs), produced by various bacterial genera make them potentially useful molecular biomarkers of bacterial communities and metabolic processes in both modern and ancient environments. Building on previous work suggesting that organisms in low-oxygen environments are important contributors to BHP production in the marine water column and that there may be physiological roles for BHPs specific to these environments, this study investigated the relationship between trends in BHP structural diversity and abundance and the genetic diversity of BHP producers for the first time in a low-oxygen environment of the Eastern Tropical North Pacific. Amplification of the hopanoid biosynthesis gene for squalene hopene cyclase (sqhC) indicated far greater genetic diversity than would be predicted by examining BHP structural diversity alone and that greater sqhC genetic diversity exists in the marine environment than is represented by cultured representatives and most marine metagenomes. In addition, the genetic relationships in this data set suggest microaerophilic environments as potential “hot spots” of BHP production. Finally, structural analysis of BHPs showed that an isomer of the commonly observed BHP bacteriohopanetetrol may be linked to a producer that is more abundant in low-oxygen environments. Results of this study increase the known diversity of BHP producers and provide a detailed phylogeny with implications for the role of hopanoids in modern bacteria, as well as the evolutionary history of hopanoid biosynthesis, both of which are important considerations for future interpretations of the marine sedimentary record.     

Distribution and Life Strategies of Two Bacterial Populations in a Eutrophic Lake

Monoclonal antibodies and epifluorescence microscopy were used to determine the depth distribution of two indigenous bacterial populations in the stratified Lake Plußsee and characterize their life strategies. Populations of Comamonas acidovorans PX54 showed a depth distribution with maximum abundances in the oxic epilimnion, whereas Aeromonas hydrophila PU7718 showed a depth distribution with maximum abundances in the anoxic thermocline layer (metalimnion), i.e., in the water layer with the highest microbial activity. Resistance of PX54 to protist grazing and high metabolic versatility and growth rate of PU7718 were the most important life strategy traits for explaining the depth distribution of the two bacterial populations. Maximum abundance of PX54 was 16,000 cells per ml, and maximum abundance of PU7718 was 20,000 cells per ml. Determination of bacterial productivity in dilution cultures with different-size fractions of dissolved organic matter (DOM) from lake water indicates that low-molecular-weight (LMW) DOM is less bioreactive than total DOM (TDOM). The abundance and growth rate of PU7718 were highest in the TDOM fractions, whereas those of PX54 were highest in the LMW DOM fraction, demonstrating that PX54 can grow well on the less bioreactive DOM fraction. We estimated that 13 to 24% of the entire bacterial community and 14% of PU7718 were removed by viral lysis, whereas no significant effect of viral lysis on PX54 could be detected. Growth rates of PX54 (0.11 to 0.13 h⁻¹) were higher than those of the entire bacterial community (0.04 to 0.08 h⁻¹) but lower than those of PU7718 (0.26 to 0.31 h⁻¹). In undiluted cultures, the growth rates were significantly lower, pointing to density effects such as resource limitation or antibiosis, and the effects were stronger for PU7718 and the entire bacterial community than for PX54. Life strategy characterizations based on data from literature and this study revealed that the fast-growing and metabolically versatile A. hydrophila PU7718 is an r-strategist or opportunistic population in Lake Plußsee, whereas the grazing-resistant C. acidovorans PX54 is rather a K-strategist or equilibrium population.     

Chemical Detection of Microbial Prey by Bacterial Predators

A motile, predacious bacterium which degraded Pythium debaryanum was strongly attracted to substances released into the medium by the fungus. A nonpredacious bacterium was not attracted to these substances. The predator bacterium was specifically attracted to cellulose and its oligomers which are known to be components of the cell wall of Pythium. Ethanol inhibited chemotaxis of the bacterium without affecting either its motility or its ability to degrade cellulose. A second predacious bacterium was isolated for the alga, Skeletonema costatum. The role of chemoreception in the detection of microbial prey by bacterial predators in natural habitats is discussed.     

Chemical States of Bacterial Spores: Dry-Heat Resistance

Mature bacterial spores can be manipulated by chemical pretreatments between states sensitive and resistant to dry heat. The two chemical forms of the spore differ in dry-heat resistance by about an order of magnitude. Log survivor curves for each chemical state were approximately straight lines. The temperature dependence of dry-heat resistance for each chemical state was similar to that usually found for dry-heat resistance. A method of testing spore resistance to dry heat has been designed to minimize artifacts resulting from (i) change of chemical state during the test, (ii) effects of water vapor activity, (iii) incomplete recovery of spores from the test container and clumping of spores. Implications of the existence of different chemical resistance states for experimental strategy and testing of dry-heat resistance are discussed.