DOI: 10.5061/dryad.pc866t20s
title: Metabolic byproduct utilization and the evolution of mutually beneficial cooperation in Escherichia coli

description: Understanding how cooperation evolves in microbial populations,
 particularly under environmental stress such as antibiotic exposure,
 remains a key topic in evolutionary biology. Here, we investigate
 cooperative interactions between antibiotic-resistant and
 antibiotic-sensitive strains of Escherichia coli. Under antibiotic stress,
 a small number of antibiotic-sensitive strains rapidly evolve into
 antibiotic-resistant strains. Resistant E. coli produce indole, which
 induces a protective response in sensitive cells, enabling them to survive
 in antibiotic stress conditions. In turn, antibiotic-sensitive E. coli
 could help reduce toxic accumulation of indole, indirectly benefiting the
 resistant strain. Indole is harmful to the growth of the
 antibiotic-resistant strain but benefits the antibiotic-sensitive strain
 by helping turn-on the multi-drug exporter to neutralize the antibiotic.
 This mutual exchange leads to increased fitness for both strains in
 cocultures, demonstrating a mechanism by which mutually beneficial
 cooperation can evolve in bacterial communities. Our findings provide
 insight into how mutualism can emerge under antibiotic pressure through
 metabolic byproduct exchange, revealing new dynamics in the evolution of
 bacterial cooperation.
description: # Metabolic byproduct utilization and the evolution of mutually beneficial
 cooperation in Escherichia coli
 [https://doi.org/10.5061/dryad.pc866t20s](https://doi.org/10.5061/dryad.pc866t20s) ## Description of the data and file structure Reference Information \===================== Provenance for this README \-------------------------- \* File name: README_file.txt File/Folder Details \=================== Details for: Data.xlsx \--------------------------------------- \* Description: a comma-delimited file containing population densities, intra- and extracellular indole concentrations, and relative gene expression levels for all Escherichia coli in the study. \* Format(s): .xlsx \* Size(s): 54.3 KB Details for: Data.xlsx__Figure1 \--------------------------------------- *methods: A total of 5 µl of overnight E. coli-S culture (OD600 ~ 1.0), grown from a single colony, was inoculated to 5 mL LB liquid mediums at a norfloxacin concentration of either 0, 50, 100, or 150 ng mL-1. The colony-forming units per milliliter (CFUs/ml) were measured at 2-hour intervals during incubation at 37 ℃. The percentage of resistant E. coli-R cells was calculated as the number of colonies at 300 ng mL-1 norfloxacin concentrations relative to the total number of cells (plated on non-restrictive plates). \* Variables: \* Group: E. coli-S bacterial fluid incubation in LB medium with 0, 50, 100, 150 ng/ml norfloxacin, respectively. \* CFUs / ml of E. coli-S every 2hrs: E. coli-S population density after 2h incubation in LB medium with 0, 50, 100, 150 ng/ml norfloxacin, respectively. \* Incubation norfloxacin concentration: E. coli-S and E. coli-R population density after 2h incubation in LB medium with 0, 50, 100, 150 ng/ml norfloxacin, respectively. \* CFU (norfloxacin=300 ng/ml): E. coli-R population density \* CFU (norfloxacin=0 ng/ml): E. coli-R and E. coli-S population density \* The percentage of norfloxacin tolerant bacterial cells (%): E. coli-R population density divided by E. coli-S and E. coli-R population density. Details for: Data.xlsx__Figure2 \--------------------------------------- *methods: We further tested if the norfloxacin resistant E. coli-R could reversibly evolve to be antibiotic sensitive in a norfloxacin-free medium. Specifically, a 1:1,000 dilution of an overnight culture of E. coli-R (OD600 ~ 1.0) was grown in LB medium or in a low nutrition (0.05% glucose) M9 medium (6.8 g/L Na2HPO4, 3 g/L KH2PO4, 1 g/L NH4Cl, 0.5 g/L NaCl, 2 mM MgSO4·7H2O, 0.5 mM thiamine hydrochloride, 0.0025 g/L FeSO4·7H2O) at 37 ℃ for ten days. Norfloxacin was absent in both mediums. The cultures were transferred to fresh medium every 12 hours at a 1:1000 concentration. The proportion of E. coli-R was calculated every 24-hours as described above. \* Variables: \* E. coli-R incubated in LB medium without norfloxacin: E. coli-R incubated in LB medium without norfloxacin for 10 days. \* E. coli-R incubated in M9 medium (0.05% glucose) without norfloxacin: E. coli-R incubated in M9 medium without norfloxacin for 10 days. \* CFU (norfloxacin=300 ng/ml): same to Data.xlsx__Figure1 \* CFU (norfloxacin= 0 ng/ml): same to Data.xlsx__Figure1 \* The percentage of norfloxacin tolerant bacterial cells (%): calculate method same to Data.xlsx__Figure1. Details for: Data.xlsx__Figure4 & 5 \--------------------------------------- *methods: An E. coli-S colony and an E. coli-R colony were both inoculated to 5mL LB liquid medium followed by overnight incubation at 37oC. The two overnight cultures were diluted to the same value at OD600~1.0. Cocultures consisted of an E. coli-R dilution diluted to 1:10,000 and an E. coli-S dilution diluted to 1:100. In each monoculture, an isolate was diluted to 1:10,000 or 1:100 for an initial number of cells equivalent to the cocultures. Cultures were incubated in LB medium supplemented with norfloxacin (300 ng mL-1). Total cell counts (E. coli-R and E. coli-S) were obtained by subculturing on non-restrictive lysogeny broth/agar plates. E. coli-R cell counts were calculated as the number of colonies at 300 ng mL-1 norfloxacin concentrations. \* Variables: \* CFU of E. coli-R in cocultrues: population density of E. coli-R in cocultrues at different times. \* CFU of E. coli-S in cocultrues: population density of E. coli-S in cocultrues at different times. \* CFU of E. coli-R in monocultrues: population density of E. coli-R in monocultrue at different times. \* CFU of E. coli-S in monocultrues: population density of E. coli-S in monocultrue at different times.. \* Relative ratio (E. coli-R: E. coli-S) in cocultrues: relative ratio of population density in cocultrues at different times. Details for: Data.xlsx__Figure6 \--------------------------------------- *methods: Monocultures and cocultures of E. coli-R and E. coli-S cells were grown in LB medium supplemented with norfloxacin (300 ng mL-1) as described above, with CFUs for each culture measured at 6,12,18 hours post initiation. Cell cultures (500 μl) were collected at 6, 12 and 18 hours, centrifuged at 14,000 rpm for 60 minutes, and the supernatant collected for extracellular indole quantification. Extracellular indole was measured by reverse-phase HPLC using a C18 Waters Spherisorb ODS-2 column (25 cm × 4.6 mm, 3 µm) set at 40 °C and gradient elution with acetonitrile-0.1% (v/v), formic acid and H2O-0.1% (v/v) formic acid at the mobile phases at a flow rate of 1 mL min-1 (35:65 for 0-5 min, 65:35 for 5-12 min, 35:65 for 12-13 min, and 35:65 for 13-30 min). Under these conditions, the retention time for indole was 13.4 min, and the absorbance maximum was 271 nm. \* Variables: \* Standard curve of indole concentration determined by HPLC: High performance liquid chromatography standard curve for indole concentration ``` *indole concentration(mg/L): indole concentration *HPLC peak area (μAU*sec: Peak area corresponding to different concentrations of indole ``` \* Quantification of the indole concentration to be tested: ``` *group: Monocultrue of E. coli-R with 300 ng/ml norfloxacin：incubate E. coli-R in LB medium with 300 ng/ml norfloxacin Cocultrue of E. coli-R andE. coli-S with 300 ng/ml norfloxacin：incubate E. coli-R and E. coli-S (1:100) in LB medium with300 ng/ml norfloxacin Monocultrue of E. coli-S with 300 ng/ml norfloxacin：ncubate E. coli-S in LB medium with 300 ng/ml norfloxacin *HPLC Peak area(μv*s): Peak area corresponding to different concentrations of indole *Mass concentration (mg/L): indole mass concentration calculated through indole concentration standard curve *Molar concentration (μmol/L): indole molar concentration *CFUs/ml of E. coli-R: population density of E. coli-R in cocultrues at different times. ``` Details for: Data.xlsx__Figure7 & S4 \--------------------------------------- *methods: In order to detect whether E. coli-S absorbed indole secreted by E. coli-R, we measured the intracellular indole concentration of E. coli-S in cocultures. The overnight E. coli-S and E. coli-R cultures were both diluted to OD600~1.0. Cocultures grown in LB medium supplemented with norfloxacin (300 ng mL-1) consist of an E. coli-R dilution diluted 1:10,000 and an E. coli-S dilution diluted 1:100. In each monoculture, the initial number of cells should equivalent to the cocultures as describe above. And a control group was added in which E. coli-S dilution was mono-cultured in the absence of norfloxacin LB medium. Co-culture and mono-culture solutions grown in LB medium for 12 hours were frozen in -20℃ for future assay. In order to ensure the amount of indole absorbed by the E. coli-R in the cocultures equaled that in the monocultures, the densities of E. coli-R in both co-culture solution and mono-culture solution were equal. Similarly, the density of E. coli-S in monoculture without added norfloxacin equaled that of the E. coli-S in coculture. The amount of intracellular indole of E. coli-S in cocultures was obtained by subtracting the amount of intracellular of E. coli-R in monoculture from the total amount of intracellular indole of E. coli-R and E. coli-S in cocultures. By comparing the intracellular indole concentration of E. coli-S in cocultures under norfloxacin pressure with that of E. coli-S in monoculture without norfloxacin, it was possible to determine whether E. coli-S absorbed more indole when antibiotics were present. For the intracellular indole concentrations, E. coli cells were completely lysed with 1% SDS and 0.2% NaOH by thorough mixing. Cells were subsequently quantified by reverse-phase HPLC as described as above. \* Variables: ``` \* Standard curve of indole concentration determined by HPLC: similar to Data.xlsx__Figure5 \* Quantification of the indole concentration to be tested: *group: Monocultrue of E. coli-R with 300 ng/ml norfloxacin：incubate E. coli-R in LB medium with 300 ng/ml norfloxacin Cocultrue of E. coli-R andE. coli-S with 300 ng/ml norfloxacin：incubate E. coli-R and E. coli-S (1:100) in LB medium with300 ng/ml norfloxacin Monocultrue of E. coli-S with 0 ng/ml norfloxacin: incubate E. coli-S in LB medium without norfloxacin. Monocultrue of E. coli-S with 100 ng/ml norfloxacin：incubate E. coli-S in LB medium with 100 ng/ml norfloxacin Monocultrue of E. coli-R with 0 ng/ml norfloxacin：incubate E. coli-S in LB medium without norfloxacin *Peak area(μv*s): same to Data.xlsx__Figure5 *Mass concentration (mg/L): same to Data.xlsx__Figure5 *Molar concentration (μmol/L): same to Data.xlsx__Figure5 *CFUs/ml (*10^8) of E. coli-R or E. coli-S: population density of E. coli-R or E. coli-S in cocultrues or cocultrues. ``` Details for: Data.xlsx__Figure8 \--------------------------------------- *methods: Indole was dissolved in methanol to a concentration of 50 mmol L-1. Same norfloxacin concentrations (300 ng mL-1) were added to 5 mL of LB liquid medium with or without the presence of 500 µmol L-1 indole. 5 μl of E. coli-S (OD600~1.0) were inoculated to 5 mL of LB liquid medium with or without the presence of 500 µmol L-1 indole. 5 μl of E. coli-R (OD600~1.0) was also inoculated to 5mL LB liquid medium supplemented with norfloxacin at 300 ng mL-1. For each norfloxacin treatment, 0 or 500 µmol L-1 indole was added. OD600 of each replicate was measured after 12 hours of incubation at 37 ℃ and 220 rpm. \* Variables: ``` *Monoculture of E. coli-S with 300 ng/ml norfloxacin: E. coli-S incubate LB medium with 300 ng/ml nordfolxacin *Monoculture of E. coli-S with 300 ng/ml norfloxacin and 500 μM indole: E. coli-S incubate LB medium with 300 ng/ml nordfolxacin and 500 µmol L-1 indole *Monoculture of E. coli-R with 300 ng/ml norfloxacin: E. coli-R incubate LB medium with 300 ng/ml nordfolxacin *Monoculture of E. coli-R with 300 ng/ml norfloxacin and 500 μM indole: E. coli-R incubate LB medium with 300 ng/ml nordfolxacin and 500 µmol L-1 indole *E. coli population density (CFUs / ml): population density of E. coli-S or E. coli-R after 12 incubation. ``` Details for: Data.xlsx__FigureS1 \--------------------------------------- *methods: Optical density at 600nm (OD600) was measured by a spectrophotometer (Spectramax M5, Applied Biosystems) following overnight incubation with norfloxacin at a range of concentrations (0, 2, 4, 8, 16, 32, 64, 128, 256, 300, 512 ng mL-1). Replicate cultures (n = 8) with OD600 ≤ 0.001 were subcultured on nonrestrictive lysogeny broth/agar plates. The MBC was determined as the minimum norfloxacin concentration (300 ng mL-1) that killed at least 99.9% of the wild-type E. coli inoculum without norfloxacin. \* Variables: ``` *Added concentration of Nor\_ Norfloxacin addition concentration (ng / ml) *Growth of E. coli-S\_ The population density of E. coli-S after 12h incubation calculated by optical density at 600nm (OD600) or CFUs/ml *group: E. coli-S colony with 8 replicates ``` Details for: Data.xlsx__FigureS2 \--------------------------------------- *methods: Replicate pre-cultures of E. coli-R and E. coli-D were initiated from individual colonies growing on freshly streaked LB agar plates. Overnight pre-cultures were diluted to OD600~0.1, with 2 μl of this dilution placed into each well of a 96-well plate along with 198 μl LB medium and incubated for 24 h at 37ºC, 880 rpm. The optical density at 600nm (OD600) was recorded in a plate reader (Spectramax M5, Applied Biosystems) every hour. The intrinsic growth rate was determined from growth curves using the logistic equation fit calculated using the ‘Growthcurver’ package for R. \* Variables: ``` *Growth curve of E. coli-D (OD600): population density of E. coli-D every hour incubation. *Growth curve of E. coli-R (OD600): population density of E. coli-R every hour incubation. *group: E. coli-D or E. coli-R population. *growth rate: intrinsic growth rateof E. coli-D or E. coli-R population. ``` Details for: Data.xlsx__FigureS3 \--------------------------------------- *methods: An E. coli-S colony and an E. coli-R colony were both inoculated to 5mL LB liquid medium followed by overnight incubation at 37oC. The two overnight cultures were diluted to the same value at OD600~1.0. Cocultures consisted of an E. coli-R dilution diluted to 1:100 and an E. coli-S dilution diluted to 1:100. In E. coli-R monoculture, an isolate was also diluted to 1:100 for an initial number of cells equivalent to the cocultures. After 12 hours of incubation, both cocultures and monocultures were plated in LB agar medium with 300 ng mL-1 norfloxacin for 24 hours. The single colonies were then transferred to LB medium and grown to exponential phase to harvest cels for RNA extraction. \* Variables: *Sample name_Mnono-R: E.coli-R cells from monoculture; Co-R: E.coli-R cells from cocultures *Target gene name: tnaA or tnaB gene. *CT of target gene: CT value of target gene from qRT-PCR *CT of internal control gene: CT value of internal control gene from qRT-PCR *CT of mean value of internal control: Mean CT value of internal control gene from qRT-PCR *∆Ct: CT of target gene minus CT of mean value of internal control *CK ∆Ct Mean: Mean value of ∆Ct *∆∆Ct（∆Ct-CK ∆Ct Mean): ∆Ct minus mean value of ∆Ct *2-∆∆Ct: relative expression of gene Details for: Data.xlsx__FigureS5 \--------------------------------------- *methods: A 1: 1,000 dilution of an overnight culture of E. coli-S (S-300) or E. coli-R (R-300) was grown in 5 ml LB medium supplemented with 300 ng mL-1 norfloxacin, along with or without 5 μl of methanol. \* Variables: ``` *Monoculture of E. coli-S with 300 ng/ml norfloxacin: E. coli-S incubate LB medium with 300 ng/ml nordfolxacin *Monoculture of E. coli-S with 300 ng/ml norfloxacin and methanol: E. coli-S incubate LB medium with 300 ng/ml nordfolxacin and 5μl methanol *Monoculture of E. coli-R with 300 ng/ml norfloxacin: E. coli-R incubate LB medium with 300 ng/ml nordfolxacin *Monoculture of E. coli-R with 300 ng/ml norfloxacin and methanol: E. coli-R incubate LB medium with 300 ng/ml nordfolxacin and 5μl methanol *E. coli population density (CFUs / ml): population density of E. coli-S or E. coli-R after 12 incubation. ``` ## Code/software \# First, load the package in R([https://cran.r-project.org/](https://cran.r-project.org/)). library(growthcurver) \# Load the sample growth curve data provided in the Growthcurver package. \# The first column is the time in hours, and there is one column \# for each well in a 96-well plate. x &lt;- read.table("clipboard",header = T) \# Now, we'll use Growthcurver to summarize the growth curve data for the entire \# plate using the default background correction method ("min"). gc_out &lt;- SummarizeGrowthByPlate(x) \# We can look at the first few rows in the output using the head command. head(gc_out)