E.coli DH5α cell response to a sudden change in microfluidic chemical environment
05 Nov 2015-Vol. 2015, pp 3213-3216
TL;DR: The studies revealed that when E.coli DH5α cells were exposed to 0.1 mM sorbitol, they showed faster chemotaxis towards the attractant and achieved steady state by 60 min, which shows that the bacterial cells respond to change in local chemical environment is within few minutes.
Abstract: Motile bacteria respond to changing chemical environment by moving towards or away from a particular location. Bacterial migration under chemical gradient is one of the most studied areas in biomedical field. In this work we looked into how bacterial cells respond to sudden change in the microfluidic chemical environment. E.coli DH5α cells were subjected to an attractant gradient (0.1 mM sorbitol - attractant to E.coli cells) and after 120 min the same cells were exposed to an inhibitor (0.1 mM NiSO 4 ) gradient in the same microfluidic device. Our studies revealed that when the E.coli DH5α cells were exposed to 0.1 mM sorbitol, they showed faster chemotaxis towards the attractant (0.1 mM sorbitol) and achieved steady state by 60 min. When we replaced 0.1 mM sorbitol with 0.1 mM NiSO 4 in the device we found that that the E.coli DH5α cells started responding to change in chemical environment within 10 min and achieved steady state at the end of 60 min. This shows that the bacterial cells respond to change in local chemical environment is within few minutes.
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TL;DR: Pilot experiments show that vectors only persist in indigenous populations when under selection pressure, disappearing when this carbon source is removed, which could prime indigenous bacteria for degrading pollutants while providing minimal ecosystem disturbance.
Abstract: Engineering bacteria to clean-up oil spills is rapidly advancing but faces regulatory hurdles and environmental concerns. Here, we develop a new technology to harness indigenous soil microbial communities for bioremediation by flooding local populations with catabolic genes for petroleum hydrocarbon degradation. Overexpressing three enzymes (almA, xylE, p450cam) in Escherichia coli led to degradation of 60–99% of target hydrocarbon substrates. Mating experiments, fluorescence microscopy and TEM revealed indigenous bacteria could obtain these vectors from E. coli through several mechanisms of horizontal gene transfer (HGT), including conjugation and cytoplasmic exchange through nanotubes. Inoculating petroleum-polluted sediments with E. coli carrying the vector pSF-OXB15-p450camfusion showed that the E. coli cells died after five days but a variety of bacteria received and carried the vector for over 60 days after inoculation. Within 60 days, the total petroleum hydrocarbon content of the polluted soil was reduced by 46%. Pilot experiments show that vectors only persist in indigenous populations when under selection pressure, disappearing when this carbon source is removed. This approach to remediation could prime indigenous bacteria for degrading pollutants while providing minimal ecosystem disturbance.
29 citations
TL;DR: The results show that the spherical AuNPs intervenes in the themotaxis of E. coli DH5α cells and inhibits the cell migration, which may be due to decreased F-type ATP synthase activity and collapse of membrane potential by Au NPs, which, in turn, leads to decreased ATP levels.
Abstract: Bacteria responds to changing chemical and thermal environment by moving towards or away from a particular location. In this report, we looked into thermal gradient generation and response of E. coli DH5α cells to thermal gradient in the presence and in the absence of spherical gold nanoparticles (size: 15 to 22 nm) in a static microfluidic environment using a polydimethylsiloxane (PDMS) made microfluidic device. A PDMS-agarose based microfluidic device for generating thermal gradient has been developed and the thermal gradient generation in the device has been validated with the numerical simulation. Our studies revealed that the presence of gold nanoparticles, AuNPs (0.649 μg/mL) has no effect on the thermal gradient generation. The E. coli DH5α cells have been treated with AuNPs of two different concentrations (0.649 μg/mL and 0.008 μg/mL). The thermotaxis behavior of cells in the presence of AuNPs has been studied and compared to the thermotaxis of E.coli DH5α cells in the absence of AuNPs. In case of thermotaxis, in the absence of the AuNPs, the E. coli DH5α cells showed better thermotaxis towards lower temperature range, whereas in the presence of AuNPs (0.649 μg/mL and 0.008 μg/mL) thermotaxis of the E. coli DH5α cells has been inhibited. The results show that the spherical AuNPs intervenes in the themotaxis of E. coli DH5α cells and inhibits the cell migration. The reason for the failure in thermotaxis response mechanism may be due to decreased F-type ATP synthase activity and collapse of membrane potential by AuNPs, which, in turn, leads to decreased ATP levels. This has been hypothesized since both thermotaxis and chemotaxis follows the same response mechanism for migration in which ATP plays critical role.
7 citations
TL;DR: In this article, the authors developed a new technology to harness indigenous soil microbial communities for bioremediation by flooding local populations with catabolic genes for petroleum hydrocarbon degradation, which could prime indigenous bacteria for degrading pollutants.
Abstract: Engineering bacteria to clean-up oil spills is rapidly advancing but faces regulatory hurdles and environmental concerns. Here, we develop a new technology to harness indigenous soil microbial communities for bioremediation by flooding local populations with catabolic genes for petroleum hydrocarbon degradation. Overexpressing three enzymes (almA, xylE, p450cam) in E.coli led to degradation rates of 60-99% of target hydrocarbon substrates. Mating experiments, fluorescence microscopy and TEM revealed indigenous bacteria could obtain these vectors from E.coli through conjugation. Inoculating petroleum-polluted sediments from a former refinery with engineered E.coli showed that the E.coli die after five days but a variety of bacteria received and carried the vector for over 120 days after inoculation. This approach could prime indigenous bacteria for degrading pollutants while providing minimal ecosystem disturbance.
2 citations
References
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TL;DR: The human host and its microbial flora constitute a complex ecosystem whose equilibrium serves as a remarkable example of reciprocal adaptation, leading to infections as a result of proliferation of antibiotic-resistant pathogenic bacteria.
Abstract: The human host and its microbial flora constitute a complex ecosystem whose equilibrium serves as a remarkable example of reciprocal adaptation. Intestinal bacteria play an important role in the development of the immune system. The normal intestinal flora is responsible for resistance to colonization by exogenous pathogenic microorganisms. Nevertheless, it also constitutes a reservoir of potentially pathogenic bacteria in close contact with the host. These bacteria are responsible for opportunistic infections in immunocompromised hosts. The equilibrium of the flora can be upset by antibiotics, leading to infections as a result of proliferation of antibiotic-resistant pathogenic bacteria.
160 citations
TL;DR: Enteroaggregative Escherichia coli (EAEC) is a subgroup of diarrhoeagenic E. coli that during the past decade has received increasing attention as a cause of watery diarrhoea, which is often persistent.
Abstract: Enteroaggregative Escherichia coli (EAEC) is a subgroup of diarrhoeagenic E coli (DEC) that during the past decade has received increasing attention as a cause of watery diarrhoea, which is often persistent EAEC have been isolated from children and adults worldwide As well as sporadic cases, outbreaks of EAEC-caused diarrhoea have been described The definition of EAEC is the ability of the micro-organism to adhere to epithelial cells such as HEp-2 in a very characteristic ‘stacked-brick’ pattern Although many studies searching for specific virulence factor(s) unique for this category of DEC have been published it is still unknown why the EAEC cause persistent diarrhoea In addition, the aggregative property of EAEC causes a lot of problems in serotyping due to the cells auto-agglutinating The gold standard for identification of EAEC includes isolation of the agent and an adherence assay using tissue culture, viz HEp-2 cells This assay is in most cases reliable; however, emergence of ‘atypical’ EAEC has been described in several publications In addition, the HEp-2 assay is time consuming, demands a tissue culture lab and trained staff Several molecular biological assays have been described, however, none show 100 % specificity
147 citations
TL;DR: Comparison of the observed cell distribution along the gradients with predictions from an established mathematical model shows very good agreement, providing the first quantification of chemotaxis of free-swimming cells in steady nonlinear microfluidic gradients and opening the door to bacterial chemot axis studies in gradients of arbitrary shape.
Abstract: Diffusion-based microfluidic devices can generate steady, arbitrarily shaped chemical gradients without requiring fluid flow and are ideal for studying chemotaxis of free-swimming cells such as bacteria. However, if microfluidic gradient generators are to be used to systematically study bacterial chemotaxis, it is critical to evaluate their performance with actual quantitative chemotaxis tests. We characterize and compare three diffusion-based gradient generators by confocal microscopy and numerical simulations, select an optimal design and apply it to chemotaxis experiments with Escherichia coli in both linear and nonlinear gradients. Comparison of the observed cell distribution along the gradients with predictions from an established mathematical model shows very good agreement, providing the first quantification of chemotaxis of free-swimming cells in steady nonlinear microfluidic gradients and opening the door to bacterial chemotaxis studies in gradients of arbitrary shape.
144 citations
TL;DR: The components of the Escherichia coli chemosensory system have been identified and their activities characterized, but how sensory information is processed to give an integrated response remains an open question.
Abstract: The components of the Escherichia coli chemosensory system have been identified and their activities characterized, but how sensory information is processed to give an integrated response remains an open question.
113 citations
TL;DR: A quantitative characterization of the chemotactic response in terms of intrinsic cell properties is needed in order to make reliable predictions about the migratory behavior of bacteria within the environment.
Abstract: Bacterial chemotaxis, the directed movement of a cell population in response to a chemical gradient, plays a critical role in the distribution and dynamic interaction of bacterial populations in nonmixed systems. Therefore, in order to make reliable predictions about the migratory behavior of bacteria within the environment, a quantitative characterization of the chemotactic response in terms of intrinsic cell properties is needed.The design of the stopped-flow diffusion chamber (SFDC) provides a well-characterized chemical gradient and reliable method for measuring bacterial migration behavior. During flow through the chamber, a step change in chemical concentration is imposed on a uniform suspension of bacteria. Once flow is stopped, diffusion causes a transient chemical gradient to develop, and bacteria respond by forming a band of high cell density which travels toward higher concentrations of the attractant. Changes in bacterial spatial distributions observed through light scattering are recorded on photomicrographs during a 10-min period. Computer-aided image analysis converts absorbance of the photographic negatives to a digital representation of bacterial density profiles. A mathematical model (part II) is used to quantitatively characterize these observations in terms of intrinsic cell parameters: a chemotactic sensitivity coefficient, chi(0), from the aggregate cell density accumulated in the band and a random motility coefficient, mu, from population dispersion in the absence of a chemical gradient.Using the SFDC assay and an individual-cell-based mathematical model, we successfully determined values for both of these population parameters for Escherichia coli K12 responding to fucose. The values obtained were mu = 1.1 +/- 0. 4 x 10(-5) cm(2)/s and chi(o) = 8 +/- 3 +/- 10(-5) cm(2)/s. We have demonstrated a method capable of determining these parameter values from the now validated mathematical model which will be useful for predicting bacterial migration in application systems.
112 citations