Stochastic Monte Carlo Model for Simulating the Dynamic Liquid-Liquid Phase Separation in Bacterial Cells.

There is growing evidence showing that many critical biological processes are driven by biomolecule condensates through liquid-liquid phase separation (LLPS). Although the qualitative observation and description of LLPS have been well documented, quantitative simulations of the time-dependent progression of LLPS in live cells are generally lacking. In this work, we build a stochastic Monte Carlo model to simulate the dynamic LLPS process during the formation of bacterial aggresomes. We demonstrate that the size distribution of the protein condensates evolves from an exponential-like to a bimodal-like pattern, and the number of condensates increases at the beginning and then decreases after reaching a maximum. Incorporating diffusion and collision, our simplified model recapitulates the two-step LLPS process in which many smaller condensates are formed in the first step and then merged into a few larger ones. We further reveal that the condensation speed, which can be defined by the condensates formed in unit time during the first step, is mainly determined by both the collision energy barrier and the initial protein density, while the number of condensates at the equilibrium is mainly associated with the dissociation energy barrier. Moreover, the LLPS process is not sensitive to temperature changes ranging around physiological conditions. Additionally, we consider the effect of the nucleation energy barrier on LLPS. We find that a higher nucleation energy barrier brings a slower condensation speed. Overall, we simulate the spatiotemporal dynamics of the LLPS process and provide qualitative guidance for understanding the dynamics of LLPS in bacterial cells, which can faithfully recapitulate experimental observations and facilitate the design of future experimental tests.

Multi-directional Morphological Assessment of Single Bacterial Colonies Through Non-invasive Optical Imaging.

A bacterium in bacterial colony is a basal component of bacterial studies and is therefore of considerable importance. The morphological characteristics of a single colony have been widely used as the standard to identify diverse bacterial species. However, the conventional methods for obtaining morphological information, such as microscopic techniques, cannot provide tomographic views. This study utilizes spectral-domain optical coherence tomography (SD-OCT) to observe both external and internal structures for single colonies. OCT imaging provided a clear top (projection) and lateral (cross-sectional) images, which can identify distinct features in the fifteen different bacterial species examined. Through the acquired OCT images, the elevation type in each genus was confirmed, and the quantitative measurements for morphology including a diameter, height, and elevation angle were achieved. Moreover, the OCT raw data was used to build three-dimensional images to observe outer appearances in all direction. As a result, the averaged angles of 7.92°, 14.40°, 6.78°, and 11.92° of the genus Hymenobacter, Spirosoma, Bacillus, and Deinococcus, respectively, were calculated. In conclusion, we have demonstrated that OCT is a highly feasible, non-invasive method for the identification of bacterial species, providing detailed morphological and visual information. It thus has strong potential for applications in bacteriology, as well as biomedical and food sciences.

An optical detection module-based biosensor using fortified bacterial beads for soil toxicity assessment.

An optical biosensor module for soil contamination assessment is presented, employing bioluminescent bacterial bioreporters encapsulated in poly-dopamine (PD)-coated alginate microbeads. The PD-coated beads displayed improved mechanical strength and stability, but somewhat delayed responses to the inducing toxicant. Using toluene as a model soil contaminant, two bioluminescent reporter strains were employed for its detection in the ambient light-blocking, temperature-controlled biosensor module. Bioluminescence of strain TV1061 (harboring an inducible grpE::luxCDABE fusion) increased and that of strain GC2 (harboring a constitutive lac::luxCDABE fusion) decreased in the presence of increasing toluene concentrations. In the former case, a maximal effect was observed in the presence of 1% toluene. This simple optical detection biosensor module may potentially be utilized for monitoring soil contamination from areas suspected of chemical pollution such petrochemical industrial zones or petrol stations.

Computer Vision Approach for the Determination of Microbial Concentration and Growth Kinetics Using a Low Cost Sensor System.

The measurement of microbial contamination is of primary importance in different fields, from environmental monitoring to food safety and clinical analysis. Today, almost all microbiology laboratories make microbial concentration measurements using the standard Plate Count Technique (PCT), a manual method that must be performed by trained personnel. Since manual PCT analysis can result in eye fatigue and errors, in particular when hundreds of samples are processed every day, automatic colony counters have been built and are commercially available. While quick and reliable, these instruments are generally expensive, thus, portable colony counters based on smartphones have been developed and are of low cost but also not accurate as the commercial benchtop instruments. In this paper, a novel computer vision sensor system is presented that can measure the microbial concentration of a sample under test and also estimate the microbial growth kinetics by monitoring the colonies grown on a Petri dish at regular time intervals. The proposed method has been in-house validated by performing PCT analysis in parallel under the same conditions and using these results as a reference. All the measurements have been carried out in a laboratory using benchtop instruments, however, such a system can also be realized as an embedded sensor system to be deployed for microbial analysis outside a laboratory environment.

Interference Disturbance Analysis Enables Single-Cell Level Growth and Mobility Characterization for Rapid Antimicrobial Susceptibility Testing.

To support the emerging battle against antimicrobial resistance (AMR), detection methods that allow fast and accurate antimicrobial susceptibility testing (AST) are urgently needed. The early identification and application of an appropriate antibiotic treatment leads to lower mortality rates and substantial cost savings and prevents the development of resistant pathogens. In this work, we present a diffraction-based method, which is capable of quantitative bacterial growth, mobility, and susceptibility measurements. The method is based on the temporal analysis of the intensity of a light diffraction peak, which arises due to interference at a periodic pattern of gold nanostructures. The presence of bacteria disturbs the constructive interference, leading to an intensity decrease and thus allows the monitoring of bacterial growth in very low volumes. We demonstrate the direct correlation of the decrease in diffraction peak intensity with bacterial cell number starting from single cells and show the capability for rapid high-throughput AST measurements by determining the minimum inhibitory concentration for three different antimicrobials in less than 2-3 h as well as the susceptibility in less than 30-40 min. Furthermore, bacterial mobility is obtained from short-term fluctuations of the diffraction peak intensity and is shown to decrease by a factor of 3 during bacterial attachment to a surface. This multiparameter detection method allows for rapid AST of planktonic and of biofilm-forming bacterial strains in low volumes and in real-time without the need of high initial cell numbers.

A Markovian Approach towards Bacterial Size Control and Homeostasis in Anomalous Growth Processes.

Regardless of the progress achieved during recent years, the mechanisms coupling growth and division to attain cell size homeostasis in bacterial populations are still not well understood. In particular, there is a gap of knowledge about the mechanisms controlling anomalous growth events that are ubiquitous even in wild-type phenotypes. Thus, when cells exceed the doubling size the divisome dynamics sets a characteristic length scale that suggests a sizer property. Yet, it has been recently shown that the size at birth and the size increment still satisfy an adder-like correlation. Herein we propose a Markov chain model, that we complement with computational and experimental approaches, to clarify this issue. In this context, we show that classifying cells as a function of the characteristic size set by the divisome dynamics provides a compelling framework to understand size convergence, growth, and division at the large length scale, including the adaptation to, and rescue from, filamentation processes. Our results reveal the independence of size homeostasis on the division pattern of long cells and help to reconcile sizer concepts at the single cell level with an adder-like behavior at a population level.

A cell-based approach to characterize antimicrobial compounds through kinetic dose response.

The rapid spread of antibiotic resistance has created a pressing need for the development of novel drug screening platforms. Herein, we report on the use of cell-based kinetic dose response curves for small molecule characterization in antibiotic discovery efforts. Kinetically monitoring bacterial growth at sub-inhibitory concentrations of antimicrobial small molecules generates unique dose response profiles. We show that clustering of profiles by growth characteristics can classify antibiotics by mechanism of action. Furthermore, changes in growth kinetics have the potential to offer insight into the mechanistic action of novel molecules and can be used to predict off-target effects generated through structure-activity relationship studies. Kinetic dose response also allows for detection of unstable compounds early in the lead development process. We propose that this kinetic approach is a rapid and cost-effective means to gather critical information on antimicrobial small molecules during the hit selection and lead development pipeline.

In vitro toxicological assessment of iron oxide, aluminium oxide and copper nanoparticles in prokaryotic and eukaryotic cell types.

Metallic nanoparticles (NPs) have a variety of applications in different industries including pharmaceutical industry where these NPs are used mainly for image analysis and drug delivery. The increasing interest in nanotechnology is largely associated with undefined risks to the human health and to the environment. Therefore, in the present study cytotoxic and genotoxic effects of iron oxide, aluminium oxide and copper nanoparticles were evaluated using most commonly used assays i.e. Ames assay, in vitro cytotoxicity assay, micronucleus assay and comet assay. Cytotoxicity to bacterial cells was assessed in terms of colony forming units by using Escherichia coli (gram negative) and Bacillus subtilis (gram positive). Ames assay was carried out using two bacterial strains of Salmonella typhimurium TA98 and TA100. Genotoxicity of these NPs was evaluated following exposure to monkey kidney cell line, CHS-20. No cytotoxic and genotoxic effects were observed for iron oxide, and aluminium oxide NPs. Copper NPs were found mutagenic in TA98 and in TA100 and also found cytotoxic in dose dependent manner. Copper NPs induced significant (p < 0.01) increase in number of binucleated cells with micronuclei (96.6 ± 5.40) at the highest concentration (25 µg/mL). Copper NPs also induced DNA strand breaks at 10 µg/mL and oxidative DNA damage at 5 and 10 µg/mL. We consider these findings very useful in evaluating the genotoxic potential of NPs especially because of their increasing applications in human health and environment with limited knowledge of their toxicity and genotoxicity.

Polyhydroxyalkanoates, challenges and opportunities.

Microbial polyhydroxyalkanoates (PHA) have been developed as biodegradable plastics for the past many years. However, PHA still have only a very limited market. Because of the availability of large amount of shale gas, petroleum will not raise dramatically in price, this situation makes PHA less competitive compared with low cost petroleum based plastics. Therefore, two strategies have been adopted to meet this challenge: first, the development of a super PHA production strain combined with advanced fermentation processes to produce PHA at a low cost; second, the construction of functional PHA production strains with technology to control the precise structures of PHA molecules, this will allow the resulting PHA with high value added applications. The recent systems and synthetic biology approaches allow the above two strategies to be implemented. In the not so distant future, the new technology will allow PHA to be produced with a competitive price compared with petroleum-based plastics.

DnaA dynamics could be linked with fitness cost in bacteria.

Fitness cost is the measure of the metabolic burden of unneeded gene expression. It is defined as the lag in bacterial cells growth harboring unneeded genes relative to unburdened cells. Separate cells can concurrently adapt to the burden, demonstrating a decrease in or even a disappearance of the lag. The precise mechanisms of this adaptation are not clearly understood. One possibility is that an increased amount of free ribosomes "absorb" the unnecessary burden. In this work, the mechanism by which an increased concentration of ribosomes could result in faster growth and mask the unneeded gene expression burden is discussed. The initiation time of chromosome replication by the initiator protein DnaA, for which the accumulation speed depends on the ribosomes amount, is taken into account.

A cost-effective solution for the reliable determination of cell numbers of microorganisms in liquid culture.

The concentration of microorganisms in growth medium is an important parameter in microbiological research. One of the approaches to determine this parameter is based on the physical interaction of small particles with light that results in light scattering. Table-top spectrophotometers can be used to determine the scattering properties of a sample as a change in light transmission. However, a portable, reliable, and maintenance-free instrument that can be built from inexpensive parts could provide new research opportunities. In this report, we show how to build such an instrument. This instrument consists of a low power monochromatic light-emitting diode, a monolithic photodiode, and a microcontroller. We demonstrate that this instrument facilitates the precise determination of cell concentrations for the bacteria Escherichia coli and Pseudomonas aeruginosa as well as the cyanobacterium Synechocystis sp. PCC 6803 and the green alga Chlamydomonas reinhardtii.

Implementing best practices and validation of cryopreservation techniques for microorganisms.

Authentic, well preserved living organisms are basic elements for research in the life sciences and biotechnology. They are grown and utilized in laboratories around the world and are key to many research programmes, industrial processes and training courses. They are vouchers for publications and must be available for confirmation of results, further study or reinvestigation when new technologies become available. These biological resources must be maintained without change in biological resource collections. In order to achieve best practice in the maintenance and provision of biological materials for industry, research and education the appropriate standards must be followed. Cryopreservation is often the best preservation method available to achieve these aims, allowing long term, stable storage of important microorganisms. To promulgate best practice the Organisation for Economic Development and Co-operation (OECD published the best practice guidelines for BRCs. The OECD best practice consolidated the efforts of the UK National Culture Collections, the European Common Access to Biological Resources and Information (CABRI) project consortium and the World Federation for Culture Collections. The paper discusses quality management options and reviews cryopreservation of fungi, describing how the reproducibility and quality of the technique is maintained in order to retain the full potential of fungi.

Vacuum cleaner emissions as a source of indoor exposure to airborne particles and bacteria.

Vacuuming can be a source of indoor exposure to biological and nonbiological aerosols, although there are few data that describe the magnitude of emissions from the vacuum cleaner itself. We therefore sought to quantify emission rates of particles and bacteria from a large group of vacuum cleaners and investigate their potential determinants, including temperature, dust bags, exhaust filters, price, and age. Emissions of particles between 0.009 and 20 µm and bacteria were measured from 21 vacuums. Ultrafine (<100 nm) particle emission rates ranged from 4.0 × 10(6) to 1.1 × 10(11) particles min(-1). Emission of 0.54-20 µm particles ranged from 4.0 × 10(4) to 1.2 × 10(9) particles min(-1). PM(2.5) emissions were between 2.4 × 10(-1) and 5.4 × 10(3) µg min(-1). Bacteria emissions ranged from 0 to 7.4 × 10(5) bacteria min(-1) and were poorly correlated with dust bag bacteria content and particle emissions. Large variability in emission of all parameters was observed across the 21 vacuums, which was largely not attributable to the range of determinant factors we assessed. Vacuum cleaner emissions contribute to indoor exposure to nonbiological and biological aerosols when vacuuming, and this may vary markedly depending on the vacuum used.

Modeling the bacterial flagellum by an elastic network of rigid bodies.

Bacteria such as Escherichia coli propel themselves by rotating a bundle of helical filaments, each driven by a rotary motor embedded in the cell membrane. Each filament is an assembly of thousands of copies of the protein flagellin which assumes two different states. We model the filament by an elastic network of rigid bodies that form bonds with one another according to a scheme suggested by Namba and Vondervistz (1997 Q. Rev. Biophys. 30 1-65) and add additional binding sites at the inner part of the rigid body. Our model reproduces the helical parameters of the 12 possible polymorphic configurations very well. We demonstrate that its energetical ground state corresponds to the normal helical form, usually observed in nature, only when inner and outer binding sites of the rigid body have a large axial displacement. This finding correlates directly to the elongated shape of the flagellin molecule. An Ising Hamiltonian in our model directly addresses the two states of the flagellin protein. It contains an external field that represents external parameters which allow us to alter the ground state of the filament.

Force generation in bacteria without nucleotide-dependent bending of cytoskeletal filaments.

Binary cell division in bacteria occurs via the formation and subsequent contraction of a polymeric ring, the so-called Z ring, at the middle of the cell. This ring is made of filamenting temperature-sensitive Z (FtsZ) proteins and it shrinks in radius to generate a contractile radial force on the cell membrane. Although a few models have been proposed, the ring contraction mechanism still remains a mystery. The models rely on various physical properties of the FtsZ filaments, some of which have been verified through in vitro experiments and some of which remain unclear. A feature common to all these models is the hydrolysis-driven transition of FtsZ filaments from straight to curved conformations. While the intrinsic curvature of FtsZ filaments has been experimentally established beyond doubt, evidence has been mounting against the existence of any transition between the straight FtsZ-GTP and the curved FtsZ-GDP conformations. Preliminary results from our earlier work [B. Ghosh and A. Sain, Phys. Rev. Lett. 101, 178101 (2008)] indicated that hydrolysis-induced bending is not necessary for Z-ring contraction. Since then many new experimental observations have been reported on this subject and in view of these here we argue that our model appears even more plausible than before. In addition, we have explored more realistic features, such as how the length distribution of FtsZ filaments in the cytoplasm may influence the contraction dynamics, and we have also demonstrated that the Z ring retains approximately the same number of monomers, although not the same monomers, during the course of contraction as reported by fluorescence experiments.

Stochastic resonance emergence from a minimalistic behavioral rule.

Stochastic resonance (SR) is a phenomenon occurring in nonlinear systems by which the ability to process information, for instance the detection of weak signals is statistically enhanced by a non-zero level of noise. SR effects have been observed in a great variety of systems, comprising electronic circuits, optical devices, chemical reactions and neurons. In this paper we report the SR phenomena occurring in the execution of an extremely simple behavioral rule inspired from bacteria chemotaxis. The phenomena are quantitatively analyzed by using Markov chain models and Monte Carlo simulations.

[Comparative assessment of survival of probiotic microorganisms from commercial preparations under the conditions in vitro].

PURPOSE OF THE STUDY: Assessment of survival of probiotic microorganisms from commercial preparations under the conditions in vitro simulates digestion in human stomach and intestine. MATERIALS AND METHODS: Probiotic microorganisms from domestic and foreign commercial preparations were used in experiments. Study of survival of probiotic microorganisms was carried out on the model in vitro simulates the conditions of digestion in the human body. RESULTS: A significant reduction in the number of viable probiotic microorganisms in the 20 of 23 studied commercial preparations during their incubation in the model media is established. Probiotic microorganisms in the preparations Bactisubtil, Sporobacterin and Biosporin possess a good survival under the conditions in vitro. CONCLUSION: The selection of probiotics for the correction of intestinal microbiocenosis requires an individual approach that takes into account the character of relations of probiotic microorganisms and indigenous microorganisms of patient as well as the possibility of using advanced prebiotics in the combine treatment of dysbacteriosis.

A mathematical and computational approach for integrating the major sources of cell population heterogeneity.

Several approaches have been used in the past to model heterogeneity in bacterial cell populations, with each approach focusing on different source(s) of heterogeneity. However, a holistic approach that integrates all the major sources into a comprehensive framework applicable to cell populations is still lacking. In this work we present the mathematical formulation of a cell population master equation (CPME) that describes cell population dynamics and takes into account the major sources of heterogeneity, namely stochasticity in reaction, DNA-duplication, and division, as well as the random partitioning of species contents into the two daughter cells. The formulation also takes into account cell growth and respects the discrete nature of the molecular contents and cell numbers. We further develop a Monte Carlo algorithm for the simulation of the stochastic processes considered here. To benchmark our new framework, we first use it to quantify the effect of each source of heterogeneity on the intrinsic and the extrinsic phenotypic variability for the well-known two-promoter system used experimentally by Elowitz et al. (2002). We finally apply our framework to a more complicated system and demonstrate how the interplay between noisy gene expression and growth inhibition due to protein accumulation at the single cell level can result in complex behavior at the cell population level. The generality of our framework makes it suitable for studying a vast array of artificial and natural genetic networks. Using our Monte Carlo algorithm, cell population distributions can be predicted for the genetic architecture of interest, thereby quantifying the effect of stochasticity in intracellular reactions or the variability in the rate of physiological processes such as growth and division. Such in silico experiments can give insight into the behavior of cell populations and reveal the major sources contributing to cell population heterogeneity.

Life cycle assessment of high-rate anaerobic treatment, microbial fuel cells, and microbial electrolysis cells.

Existing wastewater treatment options are generally perceived as energy intensive and environmentally unfriendly. Much attention has been focused on two new approaches in the past years, (i) microbial fuel cells and (ii) microbial electrolysis cells, which directly generate electrical current or chemical products, respectively, during wastewater treatment. These systems are commonly denominated as bioelectrochemical systems, and a multitude of claims have been made in the past regarding the environmental impact of these treatment options. However, an in-depth study backing these claims has not been performed. Here, we have conducted a life cycle assessment (LCA) to compare the environmental impact of three industrial wastewater treatment options, (i) anaerobic treatment with biogas generation, (ii) a microbial fuel cell treatment, with direct electricity generation, and (iii) a microbial electrolysis cell, with hydrogen peroxide production. Our analysis showed that a microbial fuel cell does not provide a significant environmental benefit relative to the "conventional" anaerobic treatment option. However, a microbial electrolysis cell provides significant environmental benefits through the displacement of chemical production by conventional means. Provided that the target conversion level of 1000 A.m(-3) can be met, the decrease in greenhouse gas emissions and other environmentally harmful emissions (e.g., aromatic hydrocarbons) of the microbial electrolysis cell will be a key driver for the development of an industrial standard for this technology. Evidently, this assessment is highly dependent on the underlying assumptions, such as the used reactor materials and target performance. This provides a challenge and an opportunity for researchers in the field to select and develop appropriate and environmentally benign materials of construction, as well as demonstrate the required 1000 A.m(-3) performance at pilot and full scale.

Performance evaluation of low cost microbial fuel cell fabricated using earthen pot with biotic and abiotic cathode.

An attempt has been made to produce low cost MFC from the commercially available earthen pots in India, without involving any costly membrane. This MFC gave a maximum power output of 16.8 W/m(3) at a Coulombic efficiency (CE) of 31.3% with graphite plate cathode. With stainless steel mesh cathode and KMnO(4) as cathodic electrolyte the power production and CE of 70.48 W/m(3) and 64.5%, respectively, was obtained. The performance of this earthen pot MFC was evaluated with biotic and abiotic cathode. Although, biofilm formation on the cathode is observed to be helpful in enhancing power out put, the thicker biofilm on the cathode showed reduction in power. This MFC demonstrated competitive performance as compared to MFC incorporated with membrane. This low cost MFC, with total production cost of less than 1.0$, as per Indian market, demonstrated its utility as a wastewater treatment and onsite power generation device.