DOI: 10.5061/dryad.fqz612jvc
title: Data from: Phenotypic plasticity of antibiotic resistance, metabolism byproduct utilization and the evolution of mutually beneficial cooperation in Escherichia coli

description: Although tag-based donation and recognition have well explained how the
 cooperative individuals are positively assorted if the cooperative
 individuals possess some signals and are also able to detect such signals,
 an additional mechanism is required to explain why some individuals pay
 the costs of evolving such a tag that may not be rewarded subsequently,
 and how such tag-based cooperative individuals will meet other similar
 individuals with a very low mutation rate. Here, we show that many and
 even all Escherichia coli bacteria cells in the increased antibiotic
 concentration will plastically evolve to be antibiotic resistant
 individuals who could protect antibiotic sensitive strain from the attack
 of antibiotics, and the antibiotic resistant strain could reversibly
 evolve to be antibiotic sensitive in non-antibiotic supplement medium but
 in a harsher environment with low glucose. A further experiment showed
 that antibiotic-sensitive E. coli strain could in turn help
 reduce the concentration of indole produced by the resistant strain. This
 metabolic product 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 discharge the antibiotic. The utilization of
 metabolism byproduct indole produced by antibiotic-resistant cells
 benefits antibiotic-sensitive cells, while the indole-absorbing service of
 antibiotic sensitive cells unconsciously help in nullifying the indole
 side effect on antibiotic resistant strain, and a mutual benefit
 cooperation could therefore evolve.
description: # Data from: Phenotypic plasticity of antibiotic resistance, metabolism
 byproduct utilization and the evolution of mutually beneficial cooperation
 in Escherichia coli
 [https://doi.org/10.5061/dryad.fqz612jvc](https://doi.org/10.5061/dryad.fqz612jvc) \* File name: README.md \* Authors: Nan Ye \* Other contributors: Beibei Hou, Jianxiao Song, Derek W Dunn, Zhanshan (Sam) Ma, Rui-Wu Wang ## Description of the data and file structure 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): 49.6 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 \--------------------------------------- *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__Figure5 \--------------------------------------- *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__Figure6 \--------------------------------------- *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: ``` *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：ncubate E. coli-S in LB medium with 100 ng/ml 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__Figure7 \--------------------------------------- *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__FigureS5 \--------------------------------------- *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