Materials and Reagents

Remel^TM plastic Petri dishes (85 mm diameter) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: R80085 )
Sterile polystyrene spreading rods (SARSTEDT, catalog number: 86.1569.005 )
Carbenicillin (CAR100) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: CT0006B )
Colistin (CT10) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: CT0017B )
Polymyxin (PB300) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: CT0044B )
Dispenser for discs (included in the kit of each of the above mentioned test discs)
Cyanobacterial cultures of WT control and mutants to be tested
Boric acid, H₃BO₃ (Sigma-Aldrich, catalog number: B6768 )
Manganese(II) chloride tetrahydrate, MnCl₂·4H₂O (Sigma-Aldrich, catalog number: 221279 )
Zinc sulfate heptahydrate, ZnSO₄·7H₂O (Sigma-Aldrich, catalog number: Z1001 )
Sodium molybdate dehydrate, Na₂MoO₄·2H₂O (Sigma-Aldrich, catalog number: 331058 )
Cupric sulfate pentahydrate, CuSO₄·5H₂O (Thermo Fisher Scientific, Fischer Scientific, catalog number: C493-500 )
Cobalt(II) nitrate hexahydrate, Co(NO₃)₂·6H₂O (Sigma-Aldrich, catalog number: 239267 )
Sodium nitrate, NaNO₃ (Scharlab, catalog number: SO05010500 )
Magnesium sulfate heptahydrate, MgSO₄·7H₂O (Sigma-Aldrich, catalog number: 63138 )
CaCl₂·2H₂O (Scharlab, catalog number: CA01981000 )
Citric acid (Sigma-Aldrich, catalog number: 251275 )
Na₂-EDTA (Sigma-Aldrich, catalog number: 27285 )
Ferric ammonium citrate (Sigma-Aldrich, catalog number: F5879 )
Sodium carbonate, Na₂CO₃(Sigma-Aldrich, catalog number: 71345 )
di-potassium hydrogen phosphate, K₂HPO₄ (EMD Millipore, catalog number: 105104 )
Na-thiosulfate (solid) (Sigma-Aldrich, catalog number: 217263 )
Difco Bacto-agar (BD, catalog number: 214530 )
TES (Sigma-Aldrich, catalog number: T6541 )
100x BG11 without Fe, phosphate, carbonate (see Recipes)
1,000x ferric ammonium citrate (see Recipes)
1,000x Na₂CO₃ (see Recipes)
1,000x K₂HPO₄ (see Recipes)
BG11 solid agar plates (see Recipes)
1 M TES/NaOH buffer, pH 8.2 (see Recipes)

Equipment

Cell culture flasks (50-250 ml) with vented caps (TC flask T25) (SARSTEDT, catalog number: 83.3910.002 )
UV/VIS spectrophotometer (GlobalMarket, PG Instruments, model: T90+ ) for measuring the absorption of cell culture (OD₇₃₀).
Multisizer^TM Coulter Counter for counting cells (Beckman Coulter, model: Z Series Coulter Counter )
Laminar hood (Thermo Fisher Scientific, Thermo Scientific^TM, model: Heraguard^TM Eco Clean Bench )
Shaking incubator (80-120 rotations/min) with light (I_VIS ~60-100 µE m^-2 s^-1) and temperature adjusted to 30 °C (Eppendorf, model: New Brunswick^TM Innova^® 43 )

Procedure

Note: it is recommended that handling of cyanobacterial cultures is done under aseptic conditions using a sterile laminar hood.

100 ml of BG11 media are inoculated with cyanobacterial cells of WT and mutant at an initial OD₇₃₀ ~0.05 and let to grow for 2-3 days until the OD of the cultures measured with the spectrophotometer at 730 nm is between 0.4-0.8.
Count cells using the Beckman Coulter cell counter. Alternatively, if a cell counter is not available, cells can be counted using a hemocytometer.
Working under sterile conditions, dilute the cyanobacterial cultures with BG11 to 100,000 cells/ml.
Spread 1 ml of diluted cultures (containing 100,000 cells) of the WT and mutant strains on Petri dishes containing 40 ml of solid BG11 media respectively. Leave the plates open in the sterile hood until the liquid has evaporated.
One-three antibiotic disks are placed on each plate of the WT and mutant strains. Use at least three biological replicates of each strain to be tested.
Petri dishes are placed in an incubator with light and 30 °C and left to incubate (1-2 weeks) until green colonies are visible.
Measure the inhibition area around each antibiotic disc to the last mm (Figure 1). If the mutant shows a higher zone of inhibition compared with the WT, this phenotype could be the consequence of the mutation interfering with the biogenesis/function of cell wall.

Figure 1. Antibiotic sensitivity test assay. Susceptibility of the WT (A, C, E) and Δdeg (B, D, F) to the different antibiotics is shown by their zone of inhibition (ZOI). ZOI induced by Polymixin B is 0.55 (± 0.1) cm and 0.8 (± 0.08) cm in WT (A) and Δdeg mutant (B) respectively. ZOI of Carbenicillin was 1.6 (± 0.19) cm for the WT (C) and 2.4 (± 0.25) cm for Δdeg (D). Colistin affected similarly WT (E) and Δdeg (F), ZOI of 0.4 cm. The black arrows indicate the measured zone of inhibitions. Adapted from Cheregi et al. (2015).

Data analysis

The average diameters of the areas of inhibition (in cm) and standard deviations were calculated from four biological replicates. Each biological replicate was represented by 3 technical replicates; the technical replicates presented areas of inhibition that were identical. These data are presented in Table 3 and Figure 3 of Cheregi et al. (2015).

Notes

This protocol was adapted from the previously published study of Trautner and Vermaas (2013) and it was performed as in Cheregi et al. (2015). The efficiency of the antibiotic is decreasing after expiration date.

Recipes

Trace minerals (1 L)
2.86 g H₃BO₃
1.81 g MnCl₂·4H₂O
0.22 g ZnSO₄·7H₂O
0.39 g Na₂MoO₄·2H₂O
0.079 g CuSO₄·5H₂O
0.049 g Co(NO₃)₂·6H₂O
100x BG11 without Fe, phosphate, carbonate (1 L)
149.6 g NaNO₃
7.5 g MgSO₄·7H₂O
3.6 g CaCl₂·2H₂O
0.60 g citric acid (or 0.89 g Na-citrate, dihydrate)
1.12 ml 0.25 M Na₂-EDTA, pH 8.0
100 ml trace minerals
Other components
Ferric ammonium citrate, 6 mg/ml (1,000x): 600 mg per 100 ml dH₂O
Na₂CO₃ (1,000 x): 2 g Na₂CO₃ per 100 ml dH₂O
K₂HPO₄ (1,000 x): 3.05 g K₂HPO₄ per 100 ml dH₂O
BG11 liquid media (1 L)
10 ml 100x BG11 without Fe, phosphate, carbonate
1 ml 1,000x ferric ammonium citrate
1 ml 1,000x Na₂CO₃
1 ml 1,000x K₂HPO₄
BG11 solid agar plates (1 L)
For agar plates, add to the above:
10 ml 1 M TES/NaOH buffer pH 8.2 (long term storage in the fridge)
3 g Na-thiosulfate (solid)
15 g Difco Bacto-agar
Autoclave at 121 °C for 30 min
1 M TES/NaOH buffer, pH 8.2
Dissolve 229.2 gr of TES in distilled H₂O
The resulting solution should be clear
Bring the pH to 8.2 using a concentrated solution of NaOH

Acknowledgments

This work was supported by grants from the Swedish Energy Agency (to C. Funk).

References

Bauer, A. W., Kirby, W. M. M., Sherris, J. C. and Turck, M. (1966). Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 45(4): 493-496.
Cheregi, O., Miranda, H., Gröbner, G. and Funk, C. (2015). Inactivation of the Deg protease family in the cyanobacterium Synechocystis sp. PCC 6803 has impact on the outer cell layers. J Photoch Photobio B 152: 383-394.
Cheregi, O., Wagner, R. and Funk, C. (2016). Insights into the cyanobacterial Deg/HtrA proteases. Front Plant Sci 7:624.
Miranda, H., Cheregi, O., Netotea, S., Hvidsten, T. R., Moritz, T. and Funk, C. (2013). Co-expression analysis, proteomic and metabolomic study on the impact of a Deg/HtrA protease triple mutant in Synechocystis sp. PCC 6803 exposed to temperature and high light stress. J Proteomics 78: 294-311.
Tam, L. X., Aigner, H., Timmerman, E., Gevaert, K. and Funk, C. (2015). Proteomic approaches to identify substrates of the three Deg/HtrA proteases of the cyanobacterium Synechocystis sp. PCC 6803. Biochem J 468(3): 373-384.
Trautner, C. and Vermaas, W. F. (2013). The sll1951 gene encodes the surface layer protein of Synechocystis sp. strain PCC 6803. J Bacteriol 195(23): 5370-5380.