Heavy metal pollution is a worldwide problem with many associated health risks, including bone loss, kidney damage, and several forms of cancer. There is a great need of bioremediation of these toxic metals from the environment, as well as implementing a monitoring system to control the spreading pollution. This study focuses on the bioremediation potential of Rhodobacter sphaeroides in the presence of the toxic gold chloride (AuCl3). Growth characteristics of the bacterial cells exposed to a range of toxic gold concentrations were analyzed through the growth kinetics and the colony forming units under aerobic, photosynthetic, and anaerobic growth conditions. The localization of the gold particles within two cellular fractions, cytoplasm and the plasma membrane, are analyzed using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). Results of this study demonstrated the photosynthetic growth condition as best suited for the metal tolerance, compared to the aerobic and anaerobic growth conditions. Results also revealed the overall accumulation and localization of gold particles, while not different between the membrane and the cytoplasmic fractions increased at different concentrations of the gold contamination. The results of the localization under photosynthetic growth condition revealed the accumulation reached the highest very quickly, and an overall shift in localization of the gold particles from an equal distribution to an increase within the membrane fraction at the highest concentrations of gold contamination. The localization of the gold particles was validated by Transmission Electron Microscopy (TEM) where the results confirmed the increase in accumulation within the membrane, and photosynthetic membranes, of R. sphaeroides.
The definition of heavy metals has differed over the years, beginning with defining heavy metals as metals with a density of five times greater than water [
Whether essential or non-essential, heavy metals become toxic to organisms at high levels, resulting in bioaccumulation, modifications of conformational structure of nucleic acids and proteins, damage to the DNA and cell membrane, and interference with the oxidative phosphorylation and osmotic balance [
Microorganisms have a wide array of different tolerance mechanisms depending on the organism as well as the heavy metal involved; each mechanism is specific to particular metals or group of metals. The mechanism of tolerance is also attributed to the concentration of the metals. It has been found that when metals are within the nanomolar range, the main mechanism of tolerance includes the activity of the metallothioneins, a class of proteins that is involved in the uptake and transportation of metals, including zinc [
Metals are found within the environment in different concentrations. Gold averages about 1 - 5 ppt in natural water [
Different growth conditions may also affect the cell survivability in response to the toxic metal contaminants. Rhodobacter sphaeroides is a bacterium that belongs the class Alphaproteobacteria within the phylum of Proteobacteria, which contains many species that are able to survive toxic metal conditions, and has been studied previously in heavy metal resistance studies, including within silver and arsenic [
To expand this investigation, the current study is focused on the localization of the gold particles within the cellular fraction. If the localization of the particles is determined, it will provide more insight into the mechanism of gold tolerance of R. sphaeroides. The hypothesis tested in this study is the localization of the gold particles is highest within the membrane fraction of cells, including the photosynthetic membrane located in the cytoplasm. The concentration of gold particles within the membrane and the cytoplasmic fractions was determined by Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). The localization of the gold particles was examined using Transmission Electron Microscopy
The accumulation within the membrane fraction may indicate a tolerance mechanism of sequestration and detoxification in order to maintain cell survivability. The results will shed important insight whether bacterial gold resistance, particularly within R. sphaeroides, is due to either cellular adaptation or mutation-selection mechanisms.
The growth media used in this study consisted of Sistrom (SIS) media [
Rhodobacter sphaeroides 2.4.1 cells were obtained from stock cultures stored at −80˚C. The cells were streaked onto SIS minimal media and grown aerobically at 30˚C. For the photosynthetic growth condition, cells were streaked onto SIS plates and placed inside a photosynthetic box. The photosynthetic box provides constant 10 Watt light conditions, and sealing the plates provides the low oxygen content. Placing the plates in an anaerobic chamber and adding the appropriate anaerobic sachets with an indicator facilitated the anaerobic growth condition. The chamber, which was placed inside an incubator at 30˚C, was covered with aluminum foil to prevent the light penetrating the chamber.
Gold chloride, AuCl3, was purchased in powdered form from Sigma Aldrich. The stock gold solutions were made by suspending the powder into deionized water to a 1.0 mM concentration. The gold chloride concentrations further discussed were diluted from the stock concentration of 1.0 mM gold chloride to final concentrations of 0.1 µM, 0.5 µM, 1.0 µM, and 10.0 µM.
A series of growth kinetics was performed for each concentration under three different growth conditions. For the aerobic growth condition, a sample of R. sphaeroides cells was grown in liquid SIS media until the log phase of growth was obtained (0.6 - 0.8 optical density at 600 nm for R. sphaeroides). The log phase of growth has been identified as the optimal growth phase to study metal tolerance [
The samples were all inoculated with the bacterial cells and the subsequent gold concentrations on the same day, designated as “day zero”. The following day, the tubes for the 24-hour time period, “day one” were analyzed, while the rest of the time periods were left untouched. This process repeated until the 120-hour time period, “day five”. This methodology allows the samples to remain untouched for the duration of the study. Once all of the tubes were prepared for the aerobic growth condition, the samples were placed in a shaker incubator at 30˚C. The prepared samples for the photosynthetic condition were sealed tightly and placed in a photosynthetic box under a constant 10 Watt light condition. The anaerobic samples were overlaid with sterile mineral oil and then placed in an incubator at 30˚C and covered in aluminum foil to prevent light reaching the tubes. The aerobic growth condition was completed first, then the photosynthetic condition, and then finally anaerobic.
To determine the localization of the gold particles, subcellular fractionation was done following the protocol outlined in [
The previously fractioned samples were loaded into the tubes designed for the equipment within the TRIES analytical laboratory. To each 4 ml sample, 80 µl of 2% Nitric Acid was added and mixed by inversion. A standard calibration curve was made using stock concentrations. The stock solutions were made using: 0.0 ml, 0.00246 ml, 0.0049 ml, 0.0492 ml, 0.492 ml, and 4.92 ml of Au concentrate into 25 ml 2% HNO3 to make the stock concentrations of 0.0 mM, 0.0005 mM, 0.001 mM, 0.01 mM, 0.1 mM and 1.0 mM respectively. The machine was programmed to take the stock concentration readings and then the standard curve was analyzed for any outliers. If any outliers were present, the standard curve was reset. Once the standard curve was linear, the job file was set up through the Smart Analyzer Vision software. A new “method” was constructed and named (Au_only) rev_2. The plasma was turned on within the machine and left to heat up for 30 minutes. The job file was constructed with a series of steps that included: Pre-flush, Iteration One, Flush, Iteration Two, Flush, Iteration Three, Final Flush. This methodology was repeated for each of the samples. The machine calculates the total amount of gold (mg/L) in the average of the three iterations per sample, and each individual iteration for the samples. The data was stored and exported to an excel sheet for further analysis. Statistical analysis on the localization of the gold particles was completed using the repeated measures analysis [
R. sphaeroides cells were grown to the log-phase and exposed to 0.5 µM and 1.0 µM gold contamination under the aerobic and photosynthetic growth conditions. The samples were taken on ice to the Image Analysis Laboratory at the Veterinary Medicine and Biomedical Sciences College at the University of Texas A&M. The cells were pelleted and the supernatant was removed. The cells were fixed in 2% glutaraldehyde and 2.5% paraformaldehyde in a 0.1 M sodium cacodylate buffer for three hours. The cells were washed and enrobed in agar overnight. The enrobed cells were cut into 1 mm cubes and fixed onto a block for imaging. Pre and post stained blocks were imaged.
Statistical analysis on the localization of the gold particles was completed using the repeated measures analysis [
The localization of the gold particles within the subcellular (cytoplasmic and membrane) fractions can be seen in
The results of the localization of the gold particles within the aerobic condition between the cytoplasmic (orange bars) and membrane (grey bars) fractions, reveal no significant difference between the two distinct subcellular fractions. When the statistical analysis was performed between the two fractions, there was no significant differences found, except where the * denotes a significance factor of (p < 0.05). Within the aerobic growth condition, the localization of the gold particles does not show a signification shift that favors one fraction over the over, and the overall accumulation does not increase in any specific pattern.
In comparing the two growth conditions, the 0.1 µM gold contamination in the photosynthetic condition reveals an equilibrium between the two cellular fractions, as well as overall accumulation of the gold ions and gold bio-nanopar- ticles across the entire 120-hour incubation time. At the 0.5 µM gold concentration, a higher accumulation of gold particles is seen within the cytoplasmic fraction for the first 72 hours (p value < 0.0001), and an equilibrium between membrane and cytoplasm is reached at 96 hours, and then gold starts to get sequestered and/or reduced at higher concentration (p value < 0.05) in the membrane fraction. At the 1.0 µM gold concentration, cells revealed an equilibrium state of the gold particles between both cellular fractions in the first 24 hours, and then it is increasingly localized within the membrane fraction (p value < 0.0001) throughout the incubation of 120 hours. At the 10.0 µM gold concentration, gold is localized at much higher concentration in the membrane fraction throughout the 120 hours post exposure. The results of the localization of the gold particles within cells grown under photosynthetic conditions suggest the photosynthetic condition is better suited for the tolerance of the metal contamination. Cells grown under photosynthetic condition maintain high concentration gold particles within both subcellular fractions compared to the concentration of gold found in aerobically grown cells. As the concentration of the gold increases, the accumulation of the gold particles remains higher within the membrane. Cells which are grown in anaerobic plus light (photosynthetic condition), and produces light harvesting complexes embedded in the photosynthetic vesicles. These vesicles are membranous structure, and contain electron transport carriers and NADP dehydrogenases [
To better understand the tolerance mechanism of R. sphaeroides to gold, or any bacterium with metal resistance, it is important to determine both the uptake kinetics and the spatial and temporal distributions of the gold ions and gold nanoparticles within subcellular fractions of the cell. This information will provide further insight into the mechanism that may be operating within the bacterial cell. There are multiple mechanisms of heavy metal resistance in bacteria which include: active transport through efflux pumps, reduction of the metal ions, the production of an extracellular barrier, as well as intra or extracellular sequestration [
The localization of the gold nanoparticles is shown in
cellular growth and cell division, representative of the logarithmic phase of growth. This growth phase, as previously mentioned, is the ideal phase to study metal contamination within, and this has been captured in the samples analyzed using the transmission electron microscopy. The aerobically grown cells exposed to the gold contamination can be seen to have the gold particles within the cytoplasm and membrane of the cells. The photosynthetically grown cells were identified to contain the gold particles localizing within the cytoplasm and membrane fractions. The presence of a photosynthetic membrane was identified in several images taken of the photosynthetically grown samples across the gold contamination concentrations. The vesicle structures, photosynthetic membrane, can be seen inside the cytoplasm of the R. sphaeroides cells, as seen in Panels E and F. This photosynthetic membrane, as previously mentioned, has been identified as the site of metallic reduction in closely related organisms, such as R. capsulatus, and the visualization of the gold particles within the photosynthetic membrane can be seen [
If the transport system is the significant line of defense against the toxic effects of gold biosorption and bioaccumulation, the efflux pump located in the plasma membrane would play an important role in flushing out the excessive gold ions from the cytoplasm, and after a while it allows to maintain a state of equilibrium between the plasma membrane and the cytoplasm [
The first mechanism is the sequestration of heavy metals within the internal membrane layer, plasma membrane. The second mechanism includes the reduction of the metallic ions into less toxic metallic forms of bio-nanoparticles in the cytoplasm. The third mechanism is to actively transport the metallic ions into the cytoplasm, take out excessive gold ions from the cytoplasm to outside the cell, and over time an equilibrium is maintained between the membrane and the cytoplasm. The results of the Inductively Coupled Plasma suggest that the accumulation of gold into the bacterial cell increases as the incubation time increases as shown in
In conclusion, the localization of the gold particles within R. sphaeroides was found to be in the highest accumulation within the membrane fraction of the cells within the photosynthetic growth condition. The results of the TEM also validated the localization of the gold particles within the membranes of the cells. Across the growth conditions, a shift in the accumulation of particles within the cellular fractions indicates a shift in the tolerance mechanisms within the bacterial cells based on the ICP results. The tolerance mechanism that we propose based on the results of this study includes the sequestration and subsequent detoxification of the gold particles within the membrane (s) of R. sphaeroides. Upon analysis of the localization study done through ICP, it can be seen how the accumulation changes between the cytoplasmic fraction and into the membrane fraction over the course of time which would possibly indicate the initial accumulation within the cytoplasmic fraction until the effects of the gold particles become too toxic to the cell, at which time the gold particles are shuttled into the membrane to be detoxified. This mechanism of tolerance will be further studied, with a focus on the photosynthetic growth condition, to further valid and characterize the gold tolerance mechanism within Rhodobacter sphaeroides.
We thank Sam Houston State University Center for Enhancing Undergraduate Research Experiences and Creative Activities (EURECA) and College of Sciences and Engineering Technology (COSET) for 2016-FAST award to M. Choudhary. We thank College of Sciences and Engineering Technology (COSET) for summer stipend to Hannah Johnson and Department of Biological Sciences for the Joey Harrison Scholarship to Hannah Johnson. We also thank William Lutterschmidt and Rachelle Smith at the Texas Research Institute for Environmental Studies (TRIES) for the use of the analytical laboratory. We also thank the Image Analysis Laboratory at the Veterinary Medicine and Biomedical Sciences College at the University of Texas A&M for assistance in the TEM sample processing and imaging.
Johnson, H., Kafle, R.C. and Choudhary, M. (2017) Cellular Localization of Gold and Mechanisms of Gold Resistance in Rhodobacter sphaeroides. Advances in Microbiology, 7, 602- 616. https://doi.org/10.4236/aim.2017.78047