Protocol

1. Preparation of DNA probes

Biotin-labeled DNA probes were amplified by PCR. One of each pair of primers is labeled by biotin at the 5’ end. DNA probes were purified by extraction with an equal volume of 1:1 phenol/chloroform mixture. Following centrifugation at 10,000 rpm for 5 minutes, the upper layer was transferred to a microcentrifuge tube and mixed with an equal volume of chloroform to remove residual phenol. After centrifugation at 10,000 rpm for 5 minutes, the upper phase was transferred to a microcentrifuge tube. 1/10^th volume of 3 M sodium acetate (pH 5.2) and 3 volumes of ethanol were added to precipitate DNA. The mixture was incubated at -20^oC for at least 1 hour. DNA was pelleted by centrifugation at 15,000 g for 20 minutes and washed with 500 μl of ice-cold 70% ethanol. After air drying, DNA was resuspended in 30-100 μl of DNase-free water and stored at -20^oC.

2. EMSA reactions

EMSA reactions were set up by mixing 20 nM DNA probe, 1 μg salmon sperm DNA (Invitrogen) (non-specific competitor), EMSA binding buffer and increasing concentrations of protein to a final volume of 20 μl. Reactions were incubated at room temperature for 30 minutes. After adding 3 μl of loading buffer, reactions were electrophoresed on 6% native polyacrylamide gel in 0.5x TBE buffer at 120V for 1.5-2 hours at 4^oC using XCell SureLock^TM mini-cell system (Invitrogen). The gel was pre-run in 0.5x TBE buffer at 120V for 20 minutes before loading samples.

6% Native polyacrylamide gel (12 ml)

• 10x TBE 0.6 ml

• 40% acrylamide/bis solution (60:1) 1.8 ml

• 50% Glycerol 1.2 ml

• 10% (w/v) APS 100 μl

• TEMED 10 μl

• MilliQ H₂O 8.3 ml

10x EMSA Binding buffer (pH 7.5)

• HEPES 100 mM

• KCl 500 mM

• MgCl₂ 50 mM

• DTT 10 mM

0.5x TBE buffer

• Tris-HCl, pH 8.3 50 mM

• Boric acid 45 mM

• EDTA 0.5 mM

Loading buffer

• Glycerol 60%

• Bromophenol blue 0.025%

3. Electroblotting and detection

DNA in native polyacrylamide gel was transferred to nylon membrane (Whatman^® Nytran™ SuPerCharge) using XCell SureLock^TM mini-cell system (Invitrogen). Electroblotting was performed in 0.5x TBE buffer at 30 V for 1 hour. The membrane was incubated at 80^oC for 30 minutes to immobilize the DNA molecules. LightShift^TM chemiluminescent EMSA kit (Thermo Fisher Scientific) was used to detect the biotin-labelled DNA probes following the manufacturer’s instruction. The image was visualized by LAS-4000 Luminescent Imager equipped with ImageQuant™ LAS 4000 software (FujiFilm).

1. Preparation of DNA probes

Biotin-labeled DNA probes were amplified by PCR. One of each pair of primers is labeled by biotin at the 5’ end. DNA probes were purified by extraction with an equal volume of 1:1 phenol/chloroform mixture. Following centrifugation at 10,000 rpm for 5 minutes, the upper layer was transferred to a microcentrifuge tube and mixed with an equal volume of chloroform to remove residual phenol. After centrifugation at 10,000 rpm for 5 minutes, the upper phase was transferred to a microcentrifuge tube. 1/10^th volume of 3 M sodium acetate (pH 5.2) and 3 volumes of ethanol were added to precipitate DNA. The mixture was incubated at -20^oC for at least 1 hour. DNA was pelleted by centrifugation at 15,000 g for 20 minutes and washed with 500 μl of ice-cold 70% ethanol. After air drying, DNA was resuspended in 30-100 μl of DNase-free water and stored at -20^oC.

2. Production of G+A marker

G+A marker showing the sequence of DNA fragments in DNase I footprinting assays was produced by Maxam-Gilbert chemical sequencing reactions (Maxam & Gilbert, 1980). 10 μl of 20 μM biotin-labelled probe DNA was mixed with 25 μl of formic acid and incubated at 25^oC for 5 minutes. 200 μl of hydrazine stop buffer and 750 μl of ice-cold ethanol were added and incubated at -80^oC for 1 hour. DNA was pelleted by centrifugation at 15,000 g for 20 minutes and washed with 700 μl of ice-cold 70% ethanol twice. The DNA pellet was dried and resuspended in 100 μl of freshly prepared 10% piperidine. After reaction at 90^oC for 30 minutes, 10 μl of 3 M sodium acetate (pH 7.0) and 300 μl of ice-cold ethanol were added and incubated at -80^oC for 1 hour. DNA was pelleted by centrifugation at 15,000 g for 20 minutes and washed with 700 μl of ice-cold 70% ethanol twice. After air drying, the DNA pellet was resuspended in 20 μl of loading buffer and stored at -20^oC. 7 μl of G+A marker was used for each assay.

Hydrazine stop buffer

• Sodium acetate, pH 7.0 300 mM

• EDTA 0.1 mM

• yeast tRNA 20 μg/ml

Loading buffer

• Formamide 95%

• Bromophenol blue 0.05%

• EDTA 20 mM

3. DNase I footprinting assay

Reactions were set up by mixing 2 μM DNA probe, EMSA binding buffer and increasing concentrations of protein to a final volume of 50 μl. Reactions were incubated at room temperature for 30 minutes. 50 μl of cofactor solution was added and mixed gently. 0.02 U DNase I (Invitrogen) was added to digest DNA for 5 minutes at room temperature. Reactions were stopped by addition of 100 μl of DNase I stop solution. To extract DNA fragments from the reaction an equal volume of 1:1 phenol/chloroform mixture was added and centrifuged at 10,000 g for 5 minutes at 4^oC. The upper layer was transferred to a microcentrifuge tube and mixed with 1 μl of glycogen (20 mg/ml, Fermentas), 1/10^th volume of 3 M sodium acetate (pH 5.2) and three volumes of ethanol to precipitate DNA. The mixture was incubated at -20^oC for at least 1 hour. DNA was pelleted by centrifugation at 15,000 g for 20 minutes and washed with 500 μl of ice-cold 70% ethanol twice. After air drying, DNA was resuspended in 8 μl of loading buffer. DNA samples and G+A marker were incubated at 95^oC for 10 minutes and loaded onto a 6% sequencing gel. Electrophoresis was performed in 1x TBE buffer at 50W for 1.5-2 hours using Sequi-Gen^® GT cell system (Bio-rad). Before loading samples, the gel was pre-run in the same condition for at least 30 minutes. DNA was transferred from the sequencing gel to positively charged nylon membrane (Whatman^® Nytran™ SuPerCharge) by contact blotting overnight. A dry nylon membrane was carefully placed onto the gel and covered by three layers of Whatman 3MM paper. A glass plate and a weight of ~2 kg were placed on the Whatman 3MM paper. The nylon membrane was incubated at 80^oC for 30 minutes to immobilize the DNA molecules. LightShift^TM chemiluminescent EMSA kit (Thermo Fisher Scientific) was used to detect the biotin-labelled DNA probes following the manufacturer’s instruction. The image was visualized by LAS-4000 Luminescent Imager equipped with ImageQuant™ LAS 4000 software (FujiFilm).

Cofactor solution

• CaCl₂ 5 mM

• MgCl₂ 10 mM

DNase I stop solution

• NaCl 200 mM

• EDTA, pH 8.0 20 mM

• SDS 1%

6% sequencing gel (40 ml)

• 10x TBE 4.0 ml

• 40% acrylamide/bis solution (29:1) 6.0 ml

• Urea 16.8 g

• 25% APS 50 μl

• TEMED 50 μl

Loading buffer

• Formamide 95%

• Bromophenol blue 0.05%

• EDTA 20 mM

1. Start from bacterial cells stored in -80^oC freezer streaking onto LB agar plate for single colonies.

2. After 2 days incubation at 28 ^oC, pick up a single colony and inoculate into 5 ml LB broth.

3. Set up the fluctuation test with 6-8 replicates by inoculating ~500^* cells into a 25 ml tube containing 6 ml LB broth.

4. After 24 hours at 28 ^oC with orbital shaking, measure the frequency of nalidixic acid resistant (Nal^R) mutants.

o Make serial dilution to 10^-7, and then inoculate 100 ul into a LB agar plate for single colonies (or do drop counting).

o Inoculate 100 ul of 10⁰, 10^-1 and 10^-2 dilution into LB + Nal (75 ug/ml) plates

5. Count the colonies and calculate online the estimated mutation rate.

*Assuming the cell density of an overnight LB bacterial culture is 1x10⁹ cells/ml (or 1x10⁶ cells/ul), you can make a dilution to 10^-4 (~100 cells/ul) and then inoculate 5 ul of the 10^-4 dilution.

This protocol is used to measure the fitness of a P. fluorescens mutant relative to its wild-type ancestral strain when they are grown in sugar beet seedlings. The wild-type strain (SBW25-lacZ) is neutrally marked with a promoterless lacZ gene at a prophage site.

Day 1, Inoculate the two competing strains (mutant and SBW25-lacZ) into 5 ml LB broth directly from cells stored in -80^oC freezer.

Day 2, Mix 500 ul of the mutant and the competitor, and then spin down the cells with a benchtop centrifuge. After removing the supernatant, cells are re-suspended into 1 ml sterile water.

Note: if the mutant has a growth defect, it should be incubated for longer, and adjusted to the similar cell density (OD₆₀₀) as the wild-type strain, ensuring that the two strains are mixed at the initial ratio of approximate 1:1.

Count start ratio: make a serial dilution until 10^-7 and inoculate 100 ul of the 10^-5, 10^-6, and 10^-7 dilution onto a “LB + X-gal” plate. After two days incubation at 28^oC, count the number of white (mutant) and blue (competitor) colonies.

Plant sowing and inoculation: Add 100 ul of the cell suspension (ie the 10⁰) into 5 ml sterile water contained in a Petri dish. Add 10 coated sugar beet seeds to this solution and leave for 5 min before removing seeds (“hooked” tweezers are useful). Blot briefly on a towel and plant in sterile vermiculite in a 15 ml plastic tube. Cover with vermiculite so the seeds are about 5 mm down, try to make sure that the seed is centred and not against the side, which can mean they dry out. Water (but not too much) and place in a growth chamber, 20^oC, 16 hrs light cycle (but lab windowsill will do!). The seedlings are most susceptible to drying out early on, but they are also susceptible to over-watering. Water the vermiculite until the water level to ~ 8 ml marker. Then cover the tubes in a rack with cling film. The seeds will take 3 -5 days to germinate. Once the seeds are beginning to germinate remove the clingfilm and water every second day or so – you just have to keep an eye on things and avoid having them either too dry or too wet.

Two or three weeks after plant sowing, the plants can be harvested. We should prepare enough agar plates of “M9 + ½ CFC + X-gal”. At least five plates are needed if we want count bacteria from both shoot and rhizosphere of a plant. For each plant prepare two plastic tubes (20 ml) and add about 20-30 glass beads into each tube. Use a sterile scissor and tweezers to cut the shoot first (part above the vermiculite) and put the shoot into one tube. Then pour the whole contents of the plant-growing tube into a Petri dish and put rhizosphere (roots with attached vermiculite) into another plastic tube with glass beads.

Add 5 ml sterile water into the tube with rhizosphere, and 3 ml for the tube with shoot. Vortex vigorously for 1 min. Dilute the rhizosphere solution until 10^-3 and inoculate 100 ul of 10^-2 and 10^-3 dilutions into two “M9 + ½ CFC + X-Gal” plates. Dilute the shoot solution (100) until 10^-2 and inoculate 100 ul of 10⁰, 10^-1, and 10^-2into three “M9 + ½ CFC + X-Gal”. The plates are incubated at 28^oC.

Two days late check the results. Count white colonies (the tested mutant) and blue colonies (the competitor, wild type).

Population densities (N_i) for both competitors determined at time t = 0 and at t = T were used to calculate the Malthusian parameter (m_i), which is the average rate of increase: mi = ln[Ni(T) / Ni(0)]. Relative fitness can be expressed as the selection rate constant (SRC): r_ij = m_i − m_j, resulting in a fitness of zero when the mutant and the wild-type strain are equally fit. We can assume that the initial population size (N_i) at t = 0 is 500 and 5000 for the shoot and rhizosphere, respectively.

1. King’s Medium B (KB)

• Glycerol 10 g (ml)

• K₂HPO₄ 1.5 g

• MgSO₄-7H₂O 1.5 g

• Proteose peptone* 20 g

Dissolve in ~ 800 water and then top up to 1000 ml.

2. Lysogeny Broth (LB)

• Tryptone 10 g

• Yeast extract 5 g

• NaCl 10 g

Suspend the solids in ~800 ml of distilled or deionized water, and then top up to 1000 ml

3. Manitol glutamate yeast extract (MGY) medium

• Mannitol 10.0 gm

• L-glutamic acid 2.0 gm

• KH₂PO₄ 0.5 gm

• NaCl 0.2 gm

• MgSO₄.7H₂O 0.2 gm

• Yeast extract 0.25 gm

The above were dissolved in Milli-Q water to make up the volume to 1 L, and the pH was adjusted to 6.5.

4. M9 Minimal Medium:

• 5X M9 Salt 200 ml

• MgSO₄ (1 M) 2 ml

• CaCl₂ (1 M) 100 ul

• Glucose (20%) 20 ml

• NH4Cl (100 mg/ml) 10 ml

• Water 768 ml

5X M9 salts （500 ml）

• Na₂HPO₄ 16.96 g

• KH₂PO₄ 7.5 g

• NaCl 1.25 g

MgSO₄×7H₂O (1 M): 49.3 g in H2O to a final volume of 200 ml

CaCl₂×6H₂O (1 M): 43.82 g in H2O to a final volume of 200 ml

Glucose (20%): 40 g in 200 ml of H₂O (used concentration: 0.4%, 22.2 mM); subject to sterilization by filtration.

NH4Cl (100X) (100 mg/ml): 20 gram in 200 ml H₂O (used concentration: 1 mg/ml, 18.7 mM)

5. Casamino acid (CAA) medium

• Casamino acids (BD Diagnostic System) 5 g

• K₂HPO₄.3H₂O 1.18 g

• MgSO₄.7H₂O 0.25 g

Dissolve and add water to 1000 ml.

6. Glycerol Saline for storing bacterial cells in -80^oC freezer

• 300 ml H₂O

• 8.5 g NaCl (0.85%)

• Add Glycerol to one litre (about 700 ml)

Autoclave. When use, dispense 800 μl Glycerol Saline in a small tube, and add 1 ml bacteria culture and mix. Store at – 80^oC.

Biofilm formation was determined quantitatively using the method of crystal violet staining after O’Toole et al. (2011).

• For the biofilm assays conducted on 96-well microtiter plates, overnight bacterial culture was washed with sterile water twice and diluted 1:100 into fresh medium for assay.

• 100 μl aliquots of diluted culture were added onto a 96-well microtiter plate and incubated at 28^oC for 1, 2 or 3 days.

• Cells were dumped out and the wells were gently washed with water.

• 125 μl of 0.1% crystal violet (CV) were added and incubated at room temperature for 15 minutes.

• The plate was washed 3 times with water and allowed to dry overnight.

• 125 μl of 30% acetic acid was added to solubilize CV followed by incubation at room temperature for 15 minutes.

• Absorbance at 550 nm was measured using a Synergy 2 plate reader (Bio-Tek).

Biofilm assay performed in microcosms followed the same procedure with a large-scale usage of bacterial culture and reagents.

We have used this protocol for biolfilm quantification of P. fluorescens, P. syringae, P. aeruginosa and Staphylococcus aureus.

Growth kinetics of P. fluorescens strains can be determined in microtiter plates using a plate reader, such as the Synergy 2 multimode microplate reader equipped with Gen5 (v1.04.5) software (BioTek, Winooski, VT).

The key point is to ensure that strains in comparisons are physiologically equivalent. To do this, we can prepare bacterial inoculants directly using cells stored in glycerol saline at -80^oC. They were first inoculated into 5-ml LB broth and allowed to grow at 28^oC for ~12 h.

• To set up the growth experiments, the overnight cultures were adjusted to a similar optical density, and cells were pelleted by centrifugation.

• Washed once using the same amount of MSM salt solution.

• Starve the cells at 28°C for 2 h.

• Inoculate 2 ul into 200 ul of the tested medium (per well) in a 96-well microtiter plate.

• Turbidity was measured at a wavelength of 450 nm (and/or 600 nm) at 5-min intervals for up to either 48 or 72 h.

1. Grow bacterial strain to late-log phase.

2. Spin 1.5 ml for 1 min in microcentrifuge.

3. Resuspend cells in 567 µl TE buffer, 30 µl of 10% SDS, and 3 µl of 20 mg/ml proteinase K. Mix and incubate for 1 hour at 37°C. At the same time, warm the CTAB/NaCl solution in 65^oC water bath.

4. Add 100 µl of 5 M NaCl. Mix thoroughly.

5. Add 80 µl CTAB/NaCl solution. Mix. Incubate 10 min at 65°C.

6. Subsequently extract with equal volume of Phenol and Chloroform.

7. Precipitate DNA with 0.6 volume isopropanol or two times of ethanol.

CTAB/NaCl Solution (10% CTAB in 0.7 M NaCl)

Dissolve 4.1 g NaCl in 80 ml water and slowly add 10 g CTAB (Hexadecyltrimethyl ammonium bromide) while heating and stirring. If necessary, heat to 65°C to dissolve. Adjust final volume to 100 ml.

Proteinase K buffer: 10 mM CaCl₂

This protocol is used to introduce genes cloned in a suicide plasmid such as pUIC3 into P. fluorescens SBW25. The recombinant plasmid will be integrated into the SBW25 genome by homologous recombination.

1. Inoculate a loop of cells of the three parental strains from laboratory stock at -80^oC :

   • Donor, E. coli DH5a containing the pUIC3 recombinant plasmid in 5 ml LB + Tc;

   • Helper, E. coli DH5a containing pRK2013 (Tra⁺) in 5 ml LB + Km;

   • Recipient, P. fluorescens SBW25 in 5 ml LB.

2. Grow the E. coli and Pseudomonas strains overnight at 37^oC and 28^oC, respectively.

3. Next morning, heat a water bath to 45^oC. Add 500 ul of the recipient P. fluorescens SBW25 into an EP tube and heat shock it for 20 min.

4. During the 20 min incubation, add 500 ul donor and 500 ul helper into two separate EP tubes and then spin down. After removing the supernatant the donor and helper are mixed up by using 500 ul LB broth and sit on the table.

5. When heat shock is finished, spin down the recipient together with the mixed donor and helper.

6. Remove the supernatants and mix all three parental strain by using ~50 ul LB broth. Then transfer the mixed cells onto an LB plate and spread a little bit by using a bended tip.

7. Dry the plate in laminar flow when necessary. Then put the plate into 28^oC incubator.

8. Next day, transfer cells (using a bended tip) into a 20 ml plastic tube containing 3 ml sterile water (or ¼ Ringer’s solution). Vortex to resuspend the cells.

9. Inoculate bacterial solution into selective plates “LB + NF + Tc + X-Gal” (100 ul for each plate, maybe 2 to 3 plates in total).

10. Incubate the conjugation plates at 28^oC incubator. Single colonies should come out after two days incubation.

NF: Nitrofurantoin, 100 ug ml^-1 (prepare a stock solution of 100 mg ml^-1 in DMSO, but make fresh stock each time you need it).

• Grow bacteria overnight in broth or on plate – I generally streak on a plate and use 1-2 loopfuls scraped off the plate for each electroporation, otherwise use 1.5 mls of late log phase broth, centrifuged to remove supernatant. I generally do two tubes for each sample and keep one on ice after adding DNA to the other – gives you a balance tube, and provides a spare for quick repeat if cuvette arcs.

• Resuspend bacteria in 1 ml sterile, ice-cold 10% Glycerol, 1 mM HEPES (HEPES solutions need to be filter sterilised) in 1.5 ml microfuge tube.

• Keep on ice and sterile as much as possible throughout entire protocol (work in laminar flow), and put cuvettes on ice so they are chilled.

• Centrifuge at high speed for 30 seconds.

• Pour off supernatant and remove excess from pellet by tapping on tissue.

• Resuspend in 1 ml glycerol/HEPES.

• Repeat centrifugation and removing supernatant.

• For some strains, such as P. syringae, a third repetition is helpful to avoiding arcing.

• Resuspend pellet in one-two drops glycerol/HEPES (about 100 ul)

• Add 0.5-1 ul salt-free plasmid DNA (ligations must be cleaned up by precipitation)

• Transfer to cuvette and leave on ice for 5 minutes (wide-mouthed pipette tips help to avoid shearing of electrocompetent cells at this stage)

• Set up BIO-RAD electroporator (2 mm cuvette 2.5V; 1 mm cuvette 1.4-1.8V)

• Set screen to time constant.

• Place cuvette between electrodes, zap by holding down both buttons till it beeps.

• Look for an optimum time constant of 4.8 for 2 mm cuvette (less than 4.0 is not acceptable).

• If the cuvette goes pop throw it away – it is broken.

• Immediately remove cuvette and add 800 ul of SOC medium.

• Pour contents into sterile 15 ml Falcon tube or Sterilin tube and incubate at 28ºC for 2 hours for antibiotic resistance expression before plating.

• Cuvettes can be reused if rinsed with 70% ethanol.

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