Agarose-Based Model Ecosystem for Cultivating Methanotrophs in a Methane-Oxygen Counter Gradient

We wanted to design a simple, inexpensive way to grow methane oxidizing bacteria in the lab that more closely resembles the natural environment. We wanted to do this to uncover bacterial phenotypes that are missing from standard laboratory culture conditions and ultimately link these phenotypes to their genetic determinants. The gradient syringe is the simplified version of previously described methods to culture melanotroph in a methane-oxygen counter gradient.

This method doesn't require continual flows of gas substrates, which allows multiple replicates to be run in parallel. We also can perform biochemical assays directly on the bacteria cultured within the agarose. Researchers have virtually unlimited access to bacterial genome sequences, but it is still difficult to put all of this information into context.

Our findings show that it is critical to consider the environment in which a bacterium evolved to better understand the role of individual genes. We plan to use techniques like comparative metabolomics and proteomics to learn more about how melanotroph respond to their position within the methane-oxygen counter gradient. We also plans to culture multiple strains in the same gradient syringe to see how they interact in a spatially resolved context.

To begin, obtain a culture plate with methylomonas species LW13 colonies. Inoculate the colonies in six milliliters of nitrate mineral salts medium in a glass tube, seal the tube with a serum stopper and aluminum crimp seal. Add methane using a syringe to create a final atmosphere of 50%volume by volume methane in the air.

Shake this planktonic liquid culture at 200 revolutions per minute at room temperature until turbid, which takes about one day. Passage the liquid cultures at a one to 10 ratio into fresh media. Continue growing the liquid cultures of melanotroph to log phase growth, reaching an optical density at 600 nanometers of approximately 0.5.

To prepare the syringe, remove the accompanying plungers and place it in a sterile container. Attach a sterile polytetrafluoroethylene or PTFE filter tip to each syringe and place the syringe in a standard test tube rack with the tip facing down. Then mix one milliliter of the cells with five milliliters of nitrate mineral salts medium, and four milliliters of molten agarose in a sterile conical tube.

Slowly pour the agarose mixture into the syringe up to the eight milliliter mark and let it solidify. After approximately 15 minutes, cap the syringe with a sterile 20 millimeter rubber butyl stopper. Secure the stopper with lab tape and label the syringe.

Next, fill a large 60 milliliter syringe with 100%methane and attach a PTFE filter tip connected to a sterile 23 gauge needle. Pierce the rubber stopper of the syringe with the agarose mixture with the large syringe, and insert a second sterile needle as a gas outlet. Depress the plunger of the large syringe to allow 20 milliliters of 100%methane to flush through the head-space.

Remove the outlet needle when there are one to two milliliters of methane left in the syringe to prevent oxygen backflow. Incubate the syringe with agarose mixture and methane at 18 degrees Celsius. To extrude the agarose, replace the PTFE filter tip with a sterile 23 gauge needle and the rubber stopper with the supplied syringe plunger.

Slowly depress the plunger to dispense one milliliter increments into separate sterile 1.5 milliliter micro centrifuge tubes. The wild type LW13 strain formed a distinct horizontal band at a specific depth in the gradient syringe where both methane and oxygen concentrations were low. LW13 inoculated in gradient syringes showed a methane-oxygen counter gradient with methane depletion and oxygen consumption corresponding to the depth of the horizontal band.

The OAT deletion mutant of LW13 lacked the distinct horizontal band formation observed in the wild-type strain indicating the gene's role in this phenotype. To begin, obtain the extruded agarose segments from gradient syringes inoculated with either wild-type or mutant methylomonas species LW13. Add 0.75 milliliters of 0.85%sodium chloride in water to the extruded agarose sample and homogenize by vortexing.

Further dilute the sample one to 10 in a new micro centrifuge tube with 900 microliters of salt solution. Incubate the samples with three microliters of a one to one mixture of SYTO 9 and propidium iodide stains in the dark at room temperature for 15 minutes. To determine the cells per milliliter of agarose, sonicate the microsphere counting bead suspension in a water bath for five minutes.

Then, add 10 microliters of the suspension to the sample before flow cytometry analysis. Analyze samples using a flow cystometer. Compare side scatter versus forward scatter dot plots between cell-free control samples and inoculated agarose samples to draw bacterial event voltage gates that exclude background agarose particles.

To count colony-forming units within the gradient syringe, add 800 microliters of nitrate mineral salts medium to the extruded agarose segment and vortex for 10 seconds to aid in pi petting. Prepare a sterile 96-well plate with 180 microliters of nitrate mineral salts medium in each well. Add 20 microliters of diluted agarose sample to each well in the first column and mix.

Using a multi-channel pipette, serially dilute 20 microliters of samples tenfold, starting from the first row of wells. Label square grid plate containing nitrate mineral salts auger, or media of choice. Using a multi-channel pipette, spot five microliters from a column of the 96-well plate onto the auger plate.

Flow cytometry, using the extruded agarose samples, confirmed that the OAT deletion mutant of LW13 showed reduced cell growth as compared to the wild-type strain. Complementation of the mutant with the OAT gene restored cell numbers and band formation to levels similar to the wild-type, indicating the gene's specific role in band formation.