GFP selection assay
Suspensions of AF-GFP and WT strains were washed with AFM1 basal salts
and adjusted to OD600 of 1.0. The suspensions of the two
strains were mixed to obtain AF-GFP suspensions diluted with WT at a
ratio of 1 to 100. The pure AF-GFP strain and the diluted suspensions
were incubated using an initial OD600 of 0.005 in 5 mL
cultures of four different media: AFM1 with 40 µg/mL leucine and 19
µg/mL diaminopimelic acid; AFM1 with 40 µg/mL leucine, 19 µg/mL
diaminopimelic acid, and 250 µg/mL kanamycin sulfate (AFM1K); LSM4
[0.8 g/L (NH4)2SO4,
2.0 g/L MgSO4·7H2O, 0.1 g/L
K2HPO4, 0.19 g/L citric acid, 5 mL/L
trace mineral solution (MD-TMS), 40 µg/mL leucine, 19 µg/mL
diaminopimelic acid, 17.9 µg/mL
Fe2(SO4)3, and 1 g/L
lig-sulfur] adjusted to pH 1.8 using concentrated sulfuric acid with
250 µg/mL kanamycin sulfate; LSM4 adjusted to pH 5.0 using NaOH pellets
with 250 µg/mL kanamycin sulfate. While growth in AFM1 does not require
leucine or diaminopimelic acid, these components were added to eliminate
them as factors in applying selective pressure. The leucine,
diaminopimelic acid,
Fe2(SO4)3, and kanamycin
sulfate components of the media were added to the cultures immediately
before incubation to prevent premature degradation and precipitation.
The endpoints of the cultures were determined as complete oxidation of
ferrous iron for the AFM1 and AFM1K media and reaching pH <
1.5 and pH < 1.8 for the LSM4 media adjusted to pH 1.8 and 5.0
respectively. After reaching the endpoint of the first passage for each
culture, 50 µL of the culture was inoculated into 5 mL of fresh medium
for two more passages. At the end of each passage the remaining cells
were harvested by centrifugation and washed in AFM1 basal salts. The
cells were resuspended in a solution consisting of 250 µL modified 2:2
basal salts [22.5 g/L
(NH4)2SO4, 3.75 g/L
MgSO4·7H2O, and 0.75 g/L KCl] and 250
µL phosphate buffered saline at pH 7.4 and transferred to a flow
cytometry tube. 1 µL of SYTO 61 was added to the tube and the sample was
incubated for 15 minutes at room temperature protected from light.
Flow cytometry was conducted using a BD LSRII. All samples were analyzed
under identical photomultiplier tube settings: forward scatter (FSC) =
600 V, side scatter (SSC) = 250 V, fluorescein isothiocyanate (FITC) =
450 V (GFP), allophycocyanin (APC) = 450 V (SYTO 61). Event detection
thresholds were set as follows: FSC = 500 and SSC = 200 RFUs. 50,000
events were collected for each sample. Post-run analysis was conducted
using FCS Express 7 software. The gating strategy isolated cells by
first gating the forward and side scattering profiles to capture all
potential cells and remove debris. Then, these events were further gated
using the APC and FITC channels to isolate the population that had the
SYTO 61 stain. All events with values of APC and FITC >10
were determined to be either WT or AF-GFP cells (Fig. S2).