Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Podsumowanie

Bacteria may accumulate either detrimental or beneficial mutations during their lifetime. In a population of cells individuals that have accumulated beneficial mutations may rapidly outcompete their fellows. Here we present a simple procedure to visualize intraspecies competition in a bacterial cell population over time using fluorescently labeled individuals. 

Streszczenie

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a population always have accumulated mutations that are either detrimental or beneficial for the cell. If the fitness effect of a mutation provides the subpopulation with a strong selective growth advantage, the individuals of this subpopulation may rapidly outcompete and even completely eliminate their immediate fellows. Thus, small genetic changes and selection-driven accumulation of cells that have acquired beneficial mutations may lead to a complete shift of the genotype of a cell population. Here we present a procedure to monitor the rapid clonal expansion and elimination of beneficial and detrimental mutations, respectively, in a bacterial cell population over time by cocultivation of fluorescently labeled individuals of the Gram-positive model bacterium Bacillus subtilis. The method is easy to perform and very illustrative to display intraspecies competition among the individuals in a bacterial cell population.

Wprowadzenie

Soil bacteria are usually endowed with flexible regulatory networks and broad metabolic capacities. Both features enable the cells to adjust their catabolic and anabolic pathways to compete with their fellows and other microorganisms for the nutrients, which are available in a given ecological niche¹. However, if the bacteria are unable to adapt to their environment other mechanisms may account for survival of a species. Indeed, as many bacteria proliferate fast and the populations can reach high cell densities subpopulations may have spontaneously accumulated beneficial mutations that provide the cells with a selective growth advantage and therefore increase their fitness. Moreover, mutational hotspots and stress-induced adaptive mutagenesis can facilitate the evolution of a maladapted bacterium^2,3. Thus, accumulation of mutations and growth under continuous selection is the origin for the enormous microbial diversity, even within the same genus^4,5. As in nature, shaping of bacterial genomes does also occur in the laboratory due to continuous cultivation under selection. This is exemplified by the domestication of the Gram-positive bacterium B. subtilis, which is used worldwide in basic research and in industry. In the 1940s B. subtilis was treated with DNA-damaging X-rays followed by cultivation under a specific growth condition⁶. The mutations that have accumulated in the bacteria during their domestication account for the loss of many growth characteristics, i.e. the B. subtilis laboratory strain 168 lost the ability to form complex colonies^7,8.

Nowadays, for the best-studied model bacteria Escherichia coli and B. subtilis, a variety of powerful tools is available to genetically manipulate their genomes in order to address specific scientific questions. Sometimes the inactivation of a gene of interest causes a severe growth defect, which is then clearly visible on standard growth medium⁹. By contrast, mutations that cause a weak growth defect and thus only slightly affect fitness of the strain are often ignored. However, in both cases prolonged incubation and passaging of the mutant strains for several generations usually result in the accumulation of suppressor mutants that have restored the phenotype of the parent strain^2,9. The characterization of suppressor mutants and the identification of the mutations that have restored the growth defect of the parent mutant strain is a very helpful approach that allows elucidation of important and often novel cellular processes^10,11.

We are interested in the control of glutamate homeostasis in B. subtilis¹². Similar to E. coli, B. subtilis responds to perturbation of glutamate homeostasis (i.e. block in glutamate degradation²) by the accumulation of suppressor mutants. The genomic alterations in these suppressor mutants that were acquired by spontaneous mutation were shown to rapidly restore glutamate homeostasis^9,13. Therefore, it is not surprising that adaptation of B. subtilis to a specific growth condition during domestication of the bacterium is mirrored in enzyme synthesis and in the evolved enzymatic activities, which are involved in glutamate metabolism¹². It has been suggested that the lack of exogenous glutamate in the growth medium during the domestication process was the driving force for the emergence and fixation of the cryptic glutamate dehydrogenase (GDH) gudB^CR gene in the laboratory strain 168^2,14. This hypothesis is supported by our observation that the reduced amount of GDH activity in the laboratory strain provides the bacteria with a selective growth advantage when exogenous glutamate is scarce². Moreover, cultivation of a B. subtilis strain, synthesizing the GDH GudB, in the absence of exogenous glutamate results in the accumulation of suppressor mutants that have inactivated the gudB gene². Obviously, the presence of a catabolically active GDH is disadvantageous for the cell because endogenously produced glutamate that could otherwise be used for anabolism is degraded to ammonium and 2-oxoglutarate (Figure 1). By contrast, when glutamate is provided by the medium, a B. subtilis strain equipped with high-level GDH activity has a selective growth advantage over a strain that synthesizes only one functional GDH. It is reasonable to assume that high-level GDH activity allows the bacteria to utilize glutamate as a second carbon source in addition to other carbon sources provided by the medium² (see Figure 1). Thus, GDH activity strongly affects fitness of bacteria, depending on the availability of exogenous glutamate.

Here we present a very illustrative method to monitor and to visualize intraspecies competition between two B. subtilis strains that differ in a single locus on the chromosome (Figure 2). The two strains were labeled with the yfp and cfp genes encoding the fluorophores YFP and CFP, and cocultivated under different nutritional conditions. By sampling over time and by plating appropriate dilutions on agar plates the survivors in each of the cultures could be easily monitored using a common stereo fluorescence microscope. The procedure described in this paper is easy to perform and suitable to visualize the rapid clonal expansion and elimination of beneficial and detrimental mutations, respectively, in a cell population over time.

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Protokół

1. Preparation of Agar Plates, Culture Media, Cryostocks, and Precultures

1. Prepare growth media and required reagents (see table of materials and reagents).
2. Streak the B. subtilis strains (e.g. BP40 (rocG⁺ gudB^CR amyE::P_gudB-yfp) and BP52 (rocG⁺ gudB⁺ amyE::P_gudB-cfp) expressing one and two active GDHs, respectively)² that will be used in the competition experiment on SP medium agar plates to get single colonies. Incubate the plates overnight at 37 °C.
3. Take single colonies and inoculate sterile culture tubes containing 4 ml LB liquid medium. Grow the bacteria overnight at 28 °C and 220 rpm.
4. Grow overnight cultures at 28 °C to avoid that the cells lyse or sporulate.
5. Measure the optical density of the cultures at a wavelength of 600 nm (OD₆₀₀) using a standard spectrophotometer.
6. If the cultures have reached an OD₆₀₀ of 2.0, mix 0.75 ml of the cultures with 0.75 ml of a sterile 50% glycerol solution in 1.5 ml reaction tubes. The final OD₆₀₀ should be 1.0 to obtain cryostocks containing about 10⁸ cells/ml.
7. Store the tubes at -80 °C.
8. Make three precultures of each strain labeled with the cfp and yfp encoding fluorophore genes. Inoculate the precultures (sterile culture tubes containing 4 ml LB liquid medium) with 1 μl cells from -80 °C cryostocks. Incubate the cultures overnight at 28 °C and 220 rpm.

2. Cocultivation of Bacteria, Sample Collection, and Sample Storage

1. Freshly prepare 100 ml C-Glc and CE-Glc minimal medium (see table of reagents and materials), and transfer 20 ml of each medium into sterile 100 ml shake flasks.
2. Take 0.1 ml of the precultures that were grown overnight, dilute them with 0.9 ml LB medium in a 1.5 ml cuvette, and determine the OD₆₀₀.
3. For the competition experiment, take those precultures of the different strains that have a similar OD₆₀₀ between 1.0-1.5.
4. To obtain mixed cell populations, dilute the cells of the precultures that had the appropriate OD₆₀₀ to an OD₆₀₀ of 0.05 in 20 ml C-Glc and CE-Glc minimal medium supplemented in 100 ml shake flasks. For the competition experiment the two strains should be mixed in a 1:1 ratio.
5. Take 10 ml samples from the flasks, transfer them to 15 ml plastic tubes, harvest the cells by centrifugation for 10 min at 4,000 x g in a standard table top centrifuge, and discard the supernatant.
6. Resuspend the cells in 0.5 ml fresh LB medium, and transfer the cells to a sterile 1.5 ml reaction cup. Add 0.5 ml of 50% sterile glycerol, mix the suspension by rigorous vortexing, and store the samples in an -80 °C freezer until further treatment.
7. Incubate the cells that are left in the shake flasks for up to 24 hr at 37 °C and 220 rpm. Keep the shake flasks in the dark to prevent photo bleaching of the fluorophores.
8. Take further samples of 0.1 ml after 7 hr and 24 hr of growth. Measure and note the OD₆₀₀ of 1:10 dilutions (in C-Glc or CE-Glc minimal medium) of the samples.
9. Take the appropriate amount of cells from each culture to make cryostocks that have an OD₆₀₀ of 1.0. Store the samples at -80 °C until further treatment.

3. Sample Treatment, Plating, and Incubation for Quantitative Analyses

1. After collecting all samples, thaw the cryostocks, and dilute the cells in a 0.9% saline solution (see table of reagents and materials) up to 10^-3.
2. Plate 0.1 ml of the 10^-3 dilutions on SP medium agar plates and distribute the cells using sterile glass pipettes.
3. Incubate the plates overnight at 37 °C in the dark until single colonies have appeared.

4. Counting the Survivors by Stereo Fluorescence Microscopy for Quantitative Analysis

1. Divide the bottom of the agar plate with a black pen in four parts for a better orientation while counting the survivors under the stereo fluorescence microscope.
2. Place the plate upside down under the microscope and bring the colonies into focus using the cold light source.
3. Once the colonies are in focus, switch to the appropriate filter set for CFP to visualize the surviving cells of the cfp-labeled strain. Count the survivors of this strain by labeling the colonies with a pen and note the number.
4. Remove the labels with ethanol and switch to the YFP filter set to visualize the surviving cells of the yfp-labeled strain. Count the survivors again by labeling the colonies with a pen and note the number.

5. Sample Treatment and Microscopy for Semiquantitative Analyses

1. For illustrative figures, spot 10 µl of the 10^-4 dilution (approximately 100 cells) from step 3.1 on a SP agar plate (see Figure 3).
2. Incubate the plates overnight at 37 °C in the dark until single colonies have appeared.
3. Place the Petri dish without lid under the microscope and bring the spot into focus using the cold light source.
4. Take pictures of the spots for illustrative figures. Choose an appropriate exposure time for taking the pictures of the colonies with the cold light source.
5. Without moving the plate, change to the CFP filter set, adjust the exposure time and take a picture. Do the same with the YFP filter set and save the pictures for further analyses.

6. Data Analysis

1. Use software such as Excel for the quantitative data analyses. Based on the counted colonies, calculate the percentage of yellow and blue colonies with respect to the whole number of colonies, which are set to 100%.
2. Use the calculated numbers to create a stacked bar diagram (see Figure 4 and Figure 5). Use an image-processing program such as Adobe Photoshop to construct merged pictures of the pictures taken from the different spots. Alternatively, the freely available software ImageJ, downloadable from http://rsbweb.nih.gov/ij/ can be used for image processing.
3. Open the pictures from one spot that were taken with the CFP filter set and the YFP filter set. Optimize the contrast and the brightness to reduce any background fluorescence from the media.
4. Go to one of the pictures and select all. Copy the picture and paste it onto the other picture.
5. Either use the function “color dodge” to merge the pictures or overlay the fluorescent pictures using the channels tab. Merged pictures of the colonies from different media and different time points represent the growth of the different strains within the liquid culture.

7. Specific Tips: Dye Switch Experiment and Cocultivation of Isogenic Strains Labeled with cfp and yfp

The expression of either of the two fluorophore-encoding genes in B. subtilis might influence fitness and thus the growth rate of the bacteria. Therefore, it is recommended to perform the following experiments in order to exclude that the elimination of one competitor strain from the cell population during cultivation is simply due to a negative effect of the fluorophore:

1. Repeat the whole experiment and cocultivate inversely labeled strains (e.g. the earlier cfp-labeled strain is now labeled with the yfp gene and vice versa). Although inverse, the obtained results should be comparable to the previous observation that one strain has a selective growth advantage over the other strain.
2. Repeat the whole experiment and cocultivate isogenic strains labeled with yfp and cfp (e.g. BP40 (rocG⁺ gudB^CR amyE::P_gudB-yfp) and BP41 (rocG⁺ gudB^CR amyE::P_gudB-cfp)). Estimate the negative effect of either of the two fluorophores on fitness of the bacteria by following the composition of the cell population over time.

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Wyniki

The method described here was successfully applied to visualize intraspecies competition in a cell population consisting of B. subtilis strains that were labeled with the cfp and yfp genes encoding the fluorophores CFP and YFP, respectively. As shown in Figure 3, the method can be used to visualize intraspecies competition in a very illustrative manner. By spotting the samples on small areas, the clonal composition of the cell population was made visible at a glance. Although n...

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Dyskusje

Several methods have been developed to analyze competitive fitness of bacteria¹⁶. In many cases the bacteria were labeled with different antibiotic resistance cassettes¹⁷. Similar to our approach, labeling of the cells with antibiotic resistance cassettes allows the evaluation of competitive fitness of the bacteria during cocultivation under defined growth conditions. Moreover, this method can be used to determine competitive fitness of cells that differ from each other in a specific locus on the ch...

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Materiały

Name	Company	Catalog Number	Comments
(NH4)2SO4	Roth, Germany	3746
Agar	Difco, USA	214010
Ammonium ferric citrate (CAF)	Sigma-Aldrich, Germany	9714
CaCl2	Roth, Germany	5239
Glucose	Applichem, Germany	A3617
Glycerol	Roth, Germany	4043
K2HPO4•3H2O	Roth, Germany	6878
KCl	Applichem, Germany	A3582
KH2PO4	Roth, Germany	3904
KOH	Roth, Germany	6751
MgSO4•7H2O	Roth, Germany	P027
MnCl2	Roth, Germany	T881
MnSO4•4H2O	Merck Millipore, Germany	102786
NaCl	Roth, Germany	9265
Nutrient broth	Roth, Germany	X929
Potassium glutamate	Applichem, Germany	A3712
Tryptone	Roth, Germany	8952
Tryptophan	Applichem, Germany	A3445
Yeast extract	Roth, Germany	2363
1.5 ml Reaction tubes	Sarstedt, Germany	72,690,001
2.0 ml Reaction tubes	Sarstedt, Germany	72,691
15 ml Plastic tubes with screw cap	Sarstedt, Germany	62,554,001
Petri dishes	Sarstedt, Germany	82.1473
1.5 ml Polystyrene cuvettes	Sarstedt, Germany	67,742
15 ml Glass culture tubes	Brand, Germany	7790 22
100 ml Shake flasks with aluminium caps	Brand, Germany	928 24
Sterile 10 ml glass pipettes	Brand, Germany	278 23
Incubator (28 and 37 °C)	New Brunswick	M1282-0012
Standard pipette set (2-20 μl, 10-100 μl, 100-1,000 μl)	Eppendorf, Germany	4910 000.034, 4910 000.042,
Table top centrifuge for 1.5 and 2 ml reaction tubes	Thermo Scientific, Heraeus Fresco 21, Germany	75002425
Table top centrifuge for 15 ml plastic tubes	Heraeus Biofuge Primo R, Germany	75005440
Standard spectrophotometer	Amersham Biosciences Ultrospec 2100 pro, Germany	80-2112-21
Stereofluorescence microscope	Zeiss SteREO Lumar V12, Germany	495008-0009-000
Freezer (-20 and -80 °C)	-	-
Fridge (4 °C)	-	-
Autoclave	Zirbus, LTA 2x3x4, Germany	-
pH meter	pH-meter 766, Calimatic, Knick, Germany	766
Vortex	Vortex 3, IKA, Germany	3340000
Balance	CP2202S, Sartorius, Germany	replaced by
Black pen (permanent marker)	Staedler, Germany	317-9
Powerpoint program	Microsoft, USA	-
Office Excel program	Microsoft, USA	-	Program for data processing
Adobe Photoshop CS5	Adobe, USA	replaced by CS6, download	Computer program for image processing
Computer	PC or Mac	-
ZEN pro 2011 software for the stereofluorescence microscope	Zeiss, Germany	410135 1002 110	AxioCam MRc Rev. Obtained through Zeiss
Specific solution recipes
SP Medium
8 g Nutrient broth
0.25 mg MgSO4•7H2O
1 g KCl
15 g agar for solid SP medium			if required
add 1 L with H2O			autoclave for 20 min at 121 °C
1 ml CaCl2 (0.5 M), sterilized by filtration
1 ml MnCl2 (10 mM), sterilized by filtration
2 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration
LB Medium
10 g Tryptone
5 g Yeast extract
10 g NaCl
15 g agar for solid LB medium			if required
add 1 L with H2O			autoclave for 20 min at 121 °C
C-Glc Minimal Medium
200 ml 5 x C salts
10 ml L-Tryptophan (5 mg/ml), sterilized by filtration
10 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration
10 ml III’ salts
25 ml Glucose (20%)			autoclaved for 20 min at 121 °C
add 1 L with sterile H2O
CE-Glc Minimal Medium
200 ml 5 x C salts
10 ml L-Tryptophan (5 mg/ml), sterilized by filtration
10 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration
10 ml III’ salts
20 ml Glutamate (40%)
25 ml Glucose (20%), autoclaved for 20 min at 121 °C
add 1 L with sterile H2O
5 x C salts
20 g KH2PO4
80 g K2HPO4•3H2O
16.5 g (NH4)2SO4
add 1 L with sterile H2O			autoclave for 20 min at 121 °C
III’ salts
0.232 g MnSO4•4H2O
12.3 g MgSO4•7H2O
add 1 L with sterile H2O, autoclave for 20 min at 121 °C
40% Glutamate solution
200 g L-Glutamic acid
adjust the pH to 7.0 by adding approximately 80 g KOH
add 0.5 L with sterile H2O			autoclave for 20 min at 121 °C
0.9% Saline (NaCl) Solution
add 1 L with sterile H2O			autoclave for 20 min at 121 °C
50% Glycerol solution
295 ml Glycerol (87%)
add 0.5 L with sterile H2O			autoclave for 20 min at 121 °C
Bacteria (All strains are based on the Bacillus subtilis strain 168)
Bacillus subtilis BP40 (rocG+ gudBCR amyE::PgudB-yfp)	Laboratory strain collection
Bacillus subtilis BP41 (rocG+ gudBCR amyE::PgudB-cfp)
Bacillus subtilis BP52 (rocG+ gudB+ amyE::PgudB-cfp)
Bacillus subtilis BP156 (rocG+ gudB+ amyE::PgudB-yfp)