Evaluating the biocontrol potential of Canadian strain Bacillus velezensis 1B-23 via its surfactin production at various pHs and temperatures.

BACKGROUND: Microorganisms, including Bacillus species are used to help control plant pathogens, thereby reducing reliance on synthetic pesticides in agriculture. Bacillus velezensis strain 1B-23 has been shown to reduce symptoms of bacterial disease caused by Clavibacter michiganensis subsp. michiganensis in greenhouse-grown tomatoes, with in vitro studies implicating the lipopeptide surfactin as a key antimicrobial. While surfactin is known to be effective against many bacterial pathogens, it is inhibitory to a smaller proportion of fungi which nonetheless cause the majority of crop diseases. In addition, knowledge of optimal conditions for surfactin production in B. velezensis is lacking. RESULTS: Here, B. velezensis 1B-23 was shown to inhibit in vitro growth of 10 fungal strains including Candida albicans, Cochliobolus carbonum, Cryptococcus neoformans, Cylindrocarpon destructans Fusarium oxysporum, Fusarium solani, Monilinia fructicola, and Rhizoctonia solani, as well as two strains of C. michiganensis michiganensis. Three of the fungal strains (C. carbonum, C. neoformans, and M. fructicola) and the bacterial strains were also inhibited by purified surfactin (surfactin C, or [Leu7] surfactin C15) from B. velezensis 1B-23. Optimal surfactin production occurred in vitro at a relatively low temperature (16 °C) and a slightly acidic pH of 6.0. In addition to surfactin, B. velenzensis also produced macrolactins, cyclic dipeptides and minor amounts of iturins which could be responsible for the bioactivity against fungal strains which were not inhibited by purified surfactin C. CONCLUSIONS: Our study indicates that B. velezensis 1B-23 has potential as a biocontrol agent against both bacterial and fungal pathogens, and may be particularly useful in slightly acidic soils of cooler climates.

Characterization and genomic analysis of a diesel-degrading bacterium, Acinetobacter calcoaceticus CA16, isolated from Canadian soil.

BACKGROUND: With the high demand for diesel across the world, environmental decontamination from its improper usage, storage and accidental spills becomes necessary. One highly environmentally friendly and cost-effective decontamination method is to utilize diesel-degrading microbes as a means for bioremediation. Here, we present a newly isolated and identified strain of Acinetobacter calcoaceticus ('CA16') as a candidate for the bioremediation of diesel-contaminated areas. RESULTS: Acinetobacter calcoaceticus CA16 was able to survive and grow in minimal medium with diesel as the only source of carbon. We determined through metabolomics that A. calcoaceticus CA16 appears to be efficient at diesel degradation. Specifically, CA16 is able to degrade 82 to 92% of aliphatic alkane hydrocarbons (CnHn + 2; where n = 12-18) in 28 days. Several diesel-degrading genes (such as alkM and xcpR) that are present in other microbes were also found to be activated in CA16. CONCLUSIONS: The results presented here suggest that Acinetobacter strain CA16 has good potential in the bioremediation of diesel-polluted environments.

Correlating Live Cell Viability with Membrane Permeability Disruption Induced by Trivalent Chromium.

Cr(III) is often regarded as a trace essential micronutrient that can be found in many dietary supplements due to its participation in blood glucose regulation. However, increased levels of exposure have been linked to adverse health effects in living organisms. Herein, scanning electrochemical microscopy (SECM) was used to detect variation in membrane permeability of single cells (T24) resulting from exposure to a trivalent Cr-salt, CrCl3. By employing electrochemical mediators, ferrocenemethanol (FcMeOH) and ferrocenecarboxylic acid (FcCOO-), initially semipermeable and impermeable, respectively, complementary information was obtained. Three-dimensional COMSOL finite element analysis simulations were successfully used to quantify the permeability coefficients of each mediator by matching experimental and simulated results. Depending on the concentration of Cr(III) administered, three regions of membrane response were detected. Following exposure to low concentrations (up to 500 µM Cr(III)), their permeability coefficients were comparable to that of control cells, 80 µm/s for FcMeOH and 0 µm/s for FcCOO-. This was confirmed for both mediators. As the incubation concentrations were increased, the ability of FcMeOH to permeate the membrane decreased to a minimum of 17 µm/s at 7500 µM Cr(III), while FcCOO- remained impermeable. At the highest examined concentrations, both mediators were found to demonstrate increased membrane permeability. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability studies were also conducted on Cr(III)-treated T24 cells to correlate the SECM findings with the toxicity effects of the metal. The viability experiments revealed a similar concentration-dependent trend to the SECM cell membrane permeability study.

The effects of long duration chronic exposure to hexavalent chromium on single live cells interrogated by scanning electrochemical microscopy.

Chromium is a useful heavy metal which has been employed in numerous industry and house applications. However, there are several known health risks associated with its uses. Cr (VI) is a toxic heavy metal format which serves no essential biological role in humans. It has been associated with oxidative stress, cytotoxicity, and carcinogenicity. Contamination of groundwater or soil due to improper handling lead to long term environmental damage. This study explores the effects of long duration chronic exposure to Cr (VI) on live human cells. Herein, scanning electrochemical microscopy (SECM) depth scan imaging was employed to monitor the membrane permeability of single live human bladder cancer (T24) cells following incubation with various Cr (VI) concentration stimuli. SECM was used to provide insights into the long duration effects on membrane homeostasis of individual cells exposed to constant levels of Cr (VI). Further investigation of total population viability was performed by MTT assay. Dependent on the exposure time, transition between three distinct trends was observed. At short incubation times (≤1-3 h) with low concentrations of Cr (VI) (0-10 µM), membrane permeability was largely unaffected. As time increased a decrease in membrane permeability coefficient was observed, reaching a minimum at 3-6 h. Following this a dramatic increase in membrane permeability was observed as cell viability decreased. Higher concentrations were also found to accelerate the timeframe at which these trends occurred. These findings further demonstrate the strength of SECM as a bioanalytical technique for monitoring cellular homeostasis.

Mapping Cd²âº-induced membrane permeability changes of single live cells by means of scanning electrochemical microscopy.

Scanning Electrochemical Microscopy (SECM) is a powerful, non-invasive, analytical methodology that can be used to investigate live cell membrane permeability. Depth scan SECM imaging allowed for the generation of 2D current maps of live cells relative to electrode position in the x-z or y-z plane. Depending on resolution, one depth scan image can contain hundreds of probe approach curves (PACs). Individual PACs were obtained by simply extracting vertical cross-sections from the 2D image. These experimental PACs were overlaid onto theoretically generated PACs simulated at specific geometry conditions. Simulations were carried out using 3D models in COMSOL Multiphysics to determine the cell membrane permeability coefficients at different locations on the surface of the cells. Common in literature, theoretical PACs are generated using a 2D axially symmetric geometry. This saves on both compute time and memory utilization. However, due to symmetry limitations of the model, only one experimental PAC right above the cell can be matched with simulated PAC data. Full 3D models in this article were developed for the SECM system of live cells, allowing all experimental PACs over the entire cell to become usable. Cd(2+)-induced membrane permeability changes of single human bladder (T24) cells were investigated at several positions above the cell, displaced from the central axis. The experimental T24 cells under study were incubated with Cd(2+) in varying concentrations. It is experimentally observed that 50 and 100 µM Cd(2+) caused a decrease in membrane permeability, which was uniform across all locations over the cell regardless of Cd(2+) concentration. The Cd(2+) was found to have detrimental effects on the cell, with cells shrinking in size and volume, and the membrane permeability decreasing. A mapping technique for the analysis of the cell membrane permeability under the Cd(2+) stress is realized by the methodology presented.

Tracking live cell response to cadmium (II) concentrations by scanning electrochemical microscopy.

The biological chemistry of toxic heavy metals, such as Cd (II), has become an active area of research due to connections with increased oxidative stress, cytotoxicity, and human/animal carcinogenicity. In this study, scanning electrochemical microscopy (SECM) was used as a noninvasive technique to monitor membrane permeability of single live human bladder cancer cells (T24) subjected to exposure of Cd (II) at various concentrations. The addition of a membrane permeable redox mediator, ferrocenemethanol (FcMeOH), in combination with depth scan imaging provided probe approach curves (PACs) to reveal changes in membrane homeostasis. To demonstrate the strength of SECM as a bioanalytical technique for cell physiology and pathology, we tested responses of live cells after 1h incubations with various concentrations of Cd (II). For the first time, a trend in membrane permeability of Cd (II) treated live T24 cells was discovered. Dependent on the incubation concentration, the trend displayed an initial decrease in membrane permeability coefficient from 75µm/s for control cells to 25µm/s for cells incubated with 75µM Cd (II). This was followed by an eventual return to the permeability coefficient of control cells (75µm/s) with further increases in Cd (II) exposure. The cells were found to respond at as little as 10µM Cd (II) concentrations. This work further demonstrates the use of SECM as a bioanalytical technique to monitor cell physiology and topography. A greater insight into the complex mechanisms behind Cd (II) toxicity is anticipated.

A time course study of cadmium effect on membrane permeability of single human bladder cancer cells using scanning electrochemical microscopy.

Cd(2+) is carcinogenic to both humans and experimental animals. We present quantitative time-course imaging of Cd(2+)-induced variation in the membrane permeability of single live human bladder cancer cells (T24) to ferrocenemethanol using scanning electrochemical microscopy (SECM). High temporal resolution combined with non-invasive nature renders a time-lapse SECM depth scan, a promising method to quantitatively investigate the effectiveness, kinetics, and mechanism of metal ions based on the responses of single live cells in real time. Under unstimulated conditions, T24 cells have constant membrane permeability to ferrocenemethanol of approximately 5.0×10(-5) m/s. When cadmium is added in-situ to T24 cells, the membrane permeability increases up to 3.5×10(-4) m/s, allowing more flux of ferrocenemethanol to the ultramicroelectrode tip. This suggests an increased spreading between the phospholipid heads in the cytoplasmic membrane. Membrane permeability might be used as a measure to probe cell status in practical intoxication cases. The methodology reported here can be applied to many other metals and their interactions with extracellular biomolecules, leading insights into cell physiology and pathobiology.