Using Mycobacterium smegmatis as a Bioindicator for Zinc-Limited Growth Conditions in Mycobacteria

So our research concerns bacterial adaptations to zinc limitation that go beyond just zinc conservation. So we use the pathogenic bacteria, Mycobacterium tuberculosis, and the non-pathogenic, Mycobacterium smegmatis, in order to see how zinc limitation affects things like bacterial physiology and pathogenesis. Another thing that we're interested in are alternative ribosomal proteins, which are expressed by many bacteria during zinc limitation, but we still don't know that much about them.

Both pathogenic and non-pathogenic mycobacteria showed dramatic responses to zinc limitation with expression levels in hundreds of genes and proteins influenced by availability of the micronutrient zinc. Physiologically, this results in a profound morphogenesis for Mycobacterium smegmatis and significantly upregulated oxidative stress response in both Mycobacterium smegmatis and pathogenic Mycobacterium tuberculosis. This protocol addresses difficulty reproducing and standardizing conditions for zinc-limiting growth in the production of translationally active alternative ribosomes in mycobacteria.

We observed factors outside of our control, like a heavy rain event, correlating with zinc contamination and zinc limited media preparation. The bioindicator phenotype presented here ensures reproducibility of zinc-limiting growth outcomes. The quantifiable bioindicator phenotype described here allows for achieving zinc-limiting conditions without the use of chelating agents.

Therefore, it enables the standardized production of a translationally active alternative ribosomes and downstream applications such as macrophage or animal infections to study host-pathogen interactions. So our lab is working on understanding the specific functions of each alternative ribosomal protein and how the incorporation of those proteins into ribosomes could affect translation. And we're also trying to see if the results that we do see in M.smegmatis can be applied to MTB as well, and if those can be used to develop better TB treatments.

To begin, add five milliliters of 7H9-ADC medium to a 50 milliliter bioreactor tube. Then add 10 microliters of thawed glycerol stock cells of Mycobacterium smegmatis, and screw the lid onto the tube. Position the tube firmly at approximately a 45 degree angle and incubate at 37 degrees Celsius with 120 rpm for 18 hours.

After incubation, measure the optical density at 600 nanometers and confirm it is around 0.5 to 0.8. Centrifuge the culture at 3000 x g for five minutes. Discard the supernatant and resuspend the pellet in five milliliters of zinc-limited media, or ZLM.

During centrifugation, sterilize a cuvette by filling it with 70%ethanol until overflowing, and allow it to sit for 10 minutes. Then rinse the cuvette three times with ZLM. After the final centrifugation, resuspend the cell pellet in two milliliters of ZLM.

Blank the spectrophotometer using 600 microliters of ZLM in a cuvette. Transfer 600 microliters of washed cells to the sterilized cuvette and measure the absorbance at 600 nanometers. If the optical density is above one, use the given formula.

To calculate the volume of ZLM to be added to the 600 microliters of washed cells in the cuvette to obtain an optical density of one, add the calculated volume of ZLM to the cuvette. Mix well by pipetting five to 10 times and remeasure the absorbance. Next, add 50 milliliters of ZLM into the sterile, double autoclaved 250 milliliter plastic Erlenmeyer flask.

Inoculate the flasks by adding 50 microliters of the culture adjusted to an optical density of one from the cuvette. For zinc replete media, add 30 microliters of 10 millimolar zinc sulfate to achieve a final concentration of six micromolar. Incubate the culture at 37 degrees Celsius with 100 rpm shaking for three days to reach the stationary phase.

Each day of the growth, add 200 microliters of culture in a 96-well flat bottom microplate and measure the growth metrics on a monochromator microplate reader. To report the cell density obtained from a microplate reader, generate a standard curve to convert the reading from the path length using a set volume to the standard one centimeter path length. Set the excitation wavelength to 420 nanometers and emission at 475 nanometers to measure the fluorescence of cofactor F420.

Then set the excitation wavelength to 375 nanometers and the emission wavelength to 475 nanometers to measure the background fluorescence. Generate a Fluorescence Intensity Scan from excitation at 230 nanometers to 440 nanometers in five nanometer steps, with fixed emission at 475 nanometers. Using the following equation, calculate the percentage of fluorescence intensity at 375 nanometers to 420 nanometers.

After three days, to record cell length, clean a glass slide and cover slip with a lint-free wipe. Pipette a 10 microliter drop of culture onto the slide and place the cover slip. Gently press the cover slip so the culture spreads to nearly fill the entire area underneath.

Using a compound microscope with 100X oil immersion and 10X ocular objectives With camera attachment, visualize the prepared slide. Take pictures of areas with multiple cells that are in focus in plane and not moving. Using the same microscope and objective, image a calibration slide.

In the Fiji software, select the straight line tool and draw a line across the length of the calibration slide. Select Analyze, followed by Set Scale and input the known distance in unit for the scale bar. Then select global to apply the scale to all images and measurements.

Now open the image and use the straight line tool to draw a line along the cell's length to measure it. Copy the cell length data from the pop-up window into a spreadsheet or save it as a comma separated value file for statistical analysis. Wild-type cultures grown in ZLM showed lower optical density values compared to zinc replete media with delta altRP strains having even lower values.

Cell elongation was observed in ZLM wild-type cultures with an average cell length of nine micrometers, while zinc replete cells were shorter at around three micrometers. Delta altRP cells did not show significant elongation in zinc-limited conditions. Fluorescence scans showed decreased cofactor F420 fluorescence in ZLM wild-type cultures, with delta altRP strains showing much higher fluorescence variability.