Antibiotic Disc Assay for Synechocystis sp. PCC6803

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Journal of Photochemistry and Photobiology B: Biology
Nov 2015



This protocol describes how to investigate the integrity of the outer cell wall in the cyanobacterium Synechocystis sp. PCC6803 using antibiotics. It is adapted to the agar diffusion test (Bauer et al., 1966), in which filter paper discs impregnated with specified concentrations of antibiotics were placed on agar plates inoculated with bacteria. The antibiotics we tested, interfering with the biosynthesis/function of bacterial cell walls, will diffuse into the agar and produce a zone of cyanobacterial growth inhibition around the disc(s). The size of the inhibition zone reflects the sensitivity of the strain to the action of antibiotics, e.g., a mutation in a protein functioning within the cell wall or its construction would render the mutant strain more sensitive to the respective antibiotic. The method has proven to be useful for phenotyping a mutant of Synechocystis sp. PCC6803 lacking all three genes encoding Deg proteases. Deletion of these ATP-independent serine proteases was shown to have impact on the outer cell layers of Synechocystis cells (Cheregi et al., 2015).

Keywords: Cyanobacteria (蓝细菌), Cell wall (细胞壁), Deg proteases (Deg蛋白酶), Antibiotics (抗生素)


The cyanobacterium Synechocystis sp. PCC6803 (hereafter, Synechocystis 6803) is a model organism for studying the process of photosynthesis. While its genome was sequenced already in 1996, still more than 50% of its genes encode proteins with hypothetical or unknown function. The three genes slr1204 (htrA), sll1679 (hhoA) and sll1427 (hhoB) encode serine proteases of the Deg (degradation of periplasmic proteins) family; despite detailed analyses (see Cheregi et al., 2016 and references therein) their exact subcellular localization and substrates still are enigmatic. Previous proteomic and metabolomic characterizations of single and triple deg deletion mutants performed in our lab have shown altered expression of proteins with functions in or on the outer cell layers of Synechocystis 6803 (Miranda et al., 2013; Tam et al., 2015; Cheregi et al., 2015).

The antibiotics carbenicillin, colistin and polymyxin inhibit or disrupt the bacterial cell wall and therefore can be used to test the integrity of this cellular component in mutants: polymyxin acts on the outermost lipopolysaccharide layer surrounding the cyanobacterial S-layer, carbenicillin interferes with the peptidoglycan layer and colistin acts on the plasma membrane of gram-negative bacteria (Table 1). An agar diffusion test has been developed (Bauer et al., 1966) in which filter paper discs impregnated with specified concentrations of antibiotics are placed on agar plates inoculated with bacteria. The antibiotics will diffuse from the disc into the agar and inhibit cyanobacterial growth around it. The size of this inhibition zone then reflects the sensitivity of the strain to the antibiotic. The antibiotic disc assay method was used to characterize a triple deg protease mutant, and could be used for the characterization of any cyanobacterial mutant. However, the reader should be aware of the limitations of this assay. Despite the Sll1951 protein being the main component of the outermost cell layer of cyanobacteria, called S-layer, the antibiotic disc assay only had limited effect on a sll1951 deletion mutant (Trautner and Vermaas, 2013). Though the S-layer is compromised in the sll1951 deletion mutant, the underlying layers are still intact, preventing Carbenicillin and Polymyxin B, due to their relatively high molecular masses, to penetrate into the cell.

Table 1 describes the molecular weight, the mode of action and the ordering information for the above mentioned antibiotic discs.

Table 1. Antibiotic discs used in this protocol

Materials and Reagents

  1. RemelTM plastic Petri dishes (85 mm diameter) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R80085 )
  2. Sterile polystyrene spreading rods (SARSTEDT, catalog number: 86.1569.005 )
  3. Carbenicillin (CAR100) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: CT0006B )
  4. Colistin (CT10) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: CT0017B )
  5. Polymyxin (PB300) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: CT0044B )
  6. Dispenser for discs (included in the kit of each of the above mentioned test discs)
  7. Cyanobacterial cultures of WT control and mutants to be tested
  8. Boric acid, H3BO3 (Sigma-Aldrich, catalog number: B6768 )
  9. Manganese(II) chloride tetrahydrate, MnCl2·4H2O (Sigma-Aldrich, catalog number: 221279 )
  10. Zinc sulfate heptahydrate, ZnSO4·7H2O (Sigma-Aldrich, catalog number: Z1001 )
  11. Sodium molybdate dehydrate, Na2MoO4·2H2O (Sigma-Aldrich, catalog number: 331058 )
  12. Cupric sulfate pentahydrate, CuSO4·5H2O (Thermo Fisher Scientific, Fischer Scientific, catalog number: C493-500 )
  13. Cobalt(II) nitrate hexahydrate, Co(NO3)2·6H2O (Sigma-Aldrich, catalog number: 239267 )
  14. Sodium nitrate, NaNO3 (Scharlab, catalog number: SO05010500 )
  15. Magnesium sulfate heptahydrate, MgSO4·7H2O (Sigma-Aldrich, catalog number: 63138 )
  16. CaCl2·2H2O (Scharlab, catalog number: CA01981000 )
  17. Citric acid (Sigma-Aldrich, catalog number: 251275 )
  18. Na2-EDTA (Sigma-Aldrich, catalog number: 27285 )
  19. Ferric ammonium citrate (Sigma-Aldrich, catalog number: F5879 )
  20. Sodium carbonate, Na2CO3(Sigma-Aldrich, catalog number: 71345 )
  21. di-potassium hydrogen phosphate, K2HPO4 (EMD Millipore, catalog number: 105104 )
  22. Na-thiosulfate (solid) (Sigma-Aldrich, catalog number: 217263 )
  23. Difco Bacto-agar (BD, catalog number: 214530 )
  24. TES (Sigma-Aldrich, catalog number: T6541 )
  25. 100x BG11 without Fe, phosphate, carbonate (see Recipes)
  26. 1,000x ferric ammonium citrate (see Recipes)
  27. 1,000x Na2CO3 (see Recipes)
  28. 1,000x K2HPO4 (see Recipes)
  29. BG11 solid agar plates (see Recipes)
  30. 1 M TES/NaOH buffer, pH 8.2 (see Recipes)


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


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

  1. 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.
  2. Count cells using the Beckman Coulter cell counter. Alternatively, if a cell counter is not available, cells can be counted using a hemocytometer.
  3. Working under sterile conditions, dilute the cyanobacterial cultures with BG11 to 100,000 cells/ml.
  4. 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.
  5. 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.
  6. Petri dishes are placed in an incubator with light and 30 °C and left to incubate (1-2 weeks) until green colonies are visible.
  7. 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).


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.


  1. 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
  2. 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
  3. 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
  4. 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
  5. 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
  6. 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


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


  1. 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.
  2. 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.
  3. Cheregi, O., Wagner, R. and Funk, C. (2016). Insights into the cyanobacterial Deg/HtrA proteases. Front Plant Sci 7:624.
  4. 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.
  5. 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.
  6. 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.


该协议描述了如何研究蓝藻中的外细胞壁的完整性。 PCC6803使用抗生素。适用于琼脂扩散试验(Bauer等人,1966),其中将浸渍有特定浓度的抗生素的滤纸盘放置在接种细菌的琼脂平板上。我们测试的抗生素,干扰细菌细胞壁的生物合成/功能,将扩散到琼脂中,并在盘周围产生蓝藻生长抑制区。抑制区的大小反映了菌株对抗生素作用的敏感性,例如,在细胞壁内起作用的蛋白质的突变或其构建将使得突变菌株对相应的抗生素。已经证明该方法可用于表型集胞藻(Synechocystis)突变体的表型。 PCC6803缺乏编码Deg蛋白酶的所有三种基因。显示这些不依赖于ATP的丝氨酸蛋白酶的缺失会影响集胞藻细胞的外细胞层(Cheregi等,2015)。

背景 蓝藻属集胞藻PCC6803(以下简称集胞藻6803)是研究光合作用过程的模式生物。虽然其基因组已经在1996年进行了测序,但仍有超过50%的基因编码具有假设或未知功能的蛋白质。三个基因slr1204 ( htrA ), sll1679 hhoA )和 sll1427 编码hEB的丝氨酸蛋白酶(周质蛋白质降解)家族;尽管进行了详细的分析(参见Cheregi等人,2016和其中的参考文献),它们的准确的亚细胞定位和底物仍然是神秘的。在我们实验室中进行的单个和三重缺失突变体的先前的蛋白质组学和代谢组学表征已经显示出具有在集胞藻6803(Miranda)的外细胞层中或上方的功能的蛋白质的表达改变,2013; Tam等人,2015; Cheregi等人,2015)。


关键字:蓝细菌, 细胞壁, Deg蛋白酶, 抗生素


  1. Remel TM 塑料培养皿(直径85mm)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R80085)
  2. 无菌聚苯乙烯铺展棒(SARSTEDT,目录号:86.1569.005)
  3. 碳青霉烯(CAR100)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:CT0006B)
  4. Colistin(CT10)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:CT0017B)
  5. 多粘菌素(PB300)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:CT0044B)
  6. 光盘分配器(包含在上述每个测试光盘的套件中)
  7. WT对照的蓝藻培养物和要测试的突变体
  8. 硼酸,H 3 BO 3(Sigma-Aldrich,目录号:B6768)
  9. 锰(II)四水合物,MnCl 2·4H 2 O(Sigma-Aldrich,目录号:221279)
  10. 硫酸锌七水合物,ZnSO 4·7H 2 O(Sigma-Aldrich,目录号:Z1001)
  11. 钼酸钠脱水,Na 2 O 3 O 4·2H 2 O(Sigma-Aldrich,目录号:331058)
  12. 硫酸铜五水合物,CuSO 4·5H 2 O(Thermo Fisher Scientific,Fischer Scientific,目录号:C493-500)
  13. 硝酸钴(II)六水合物Co(NO 3 3)2·6H 2 O(Sigma-Aldrich,目录号:239267)
  14. 硝酸钠,NaNO 3(Scharlab,目录号:SO05010500)
  15. 硫酸镁七水合物,MgSO 4·7H 2 O(Sigma-Aldrich,目录号:63138)
  16. CaCl 2·2H 2 O(Scharlab,目录号:CA01981000)
  17. 柠檬酸(Sigma-Aldrich,目录号:251275)
  18. Na 2 -EDTA(Sigma-Aldrich,目录号:27285)
  19. 柠檬酸铁铵(Sigma-Aldrich,目录号:F5879)
  20. 碳酸钠,Na 2 CO 3(Sigma-Aldrich,目录号:71345)
  21. 磷酸氢二钾,K 2 HPO 4(EMD Millipore,目录号:105104)
  22. 硫代硫酸钠(固体)(Sigma-Aldrich,目录号:217263)
  23. Difco Bacto-agar(BD,目录号:214530)
  24. TES(Sigma-Aldrich,目录号:T6541)
  25. 100x BG11无Fe,磷酸盐,碳酸盐(见食谱)
  26. 1,000x柠檬酸铁铵(见食谱)
  27. 1,000x Na 2 CO 3(参见食谱)
  28. 1,000x K 2 HPO 4(见配方)
  29. BG11固体琼脂平板(参见食谱)
  30. 1 M TES/NaOH缓冲液,pH 8.2(参见食谱)


  1. 带有排气帽(TC烧瓶T25)的细胞培养瓶(50-250ml)(SARSTEDT,目录号:83.3910.002)
  2. UV/VIS分光光度计(GlobalMarket,PG Instruments,型号:T90 +),用于测量细胞培养物的吸收(OD 730)。
  3. Multisizer TM 用于计数细胞的库尔特计数器(Beckman Coulter,型号:Z Series Coulter Counter)
  4. 层层罩(Thermo Fisher Scientific,Thermo Scientific TM ,型号:Heraguard TM Eco Clean Bench)
  5. 用轻轻摇动培养箱(80-120转/分钟)(I 〜60-100μEm -2至 -1 )和温度调整到30°C(Eppendorf,型号:New Brunswick TM Innova ® 43)



  1. 将100ml BG11培养基用WT和突变体的蓝细菌细胞在初始OD 730接种,并使其生长2-3天,直到用分光光度计在730nm测量培养物的OD在0.4-0.8之间。
  2. 使用Beckman Coulter细胞计数器计数细胞。或者,如果细胞计数器不可用,则可以使用血细胞计数器计数细胞。
  3. 在无菌条件下工作,用BG11稀释蓝细菌培养物至100,000个细胞/ml
  4. 分别在含有40ml固体BG11培养基的培养皿上分散1ml稀释培养物(含有100,000个细胞)的WT和突变株。将板放在无菌罩中,直到液体蒸发。
  5. 将三分之一的抗生素盘放置在WT和突变株的每个平板上。使用每种待测试菌株的至少三种生物重复。
  6. 将培养皿放在轻度和30℃的培养箱中,并保持孵育(1-2周),直到可见绿色菌落。
  7. 测量每个抗生素盘周围的抑制区域到最后的mm(图1)。如果与WT相比,突变体显示较高的抑制区,则该表型可能是干扰细胞壁生物发生/功能的突变的结果。

    图1.抗生素敏感性试验测定 WT(A,C,E)和Δ(B,D,F)对不同抗生素的敏感性显示为它们的抑制区(ZOI)。 Polymixyn B诱导的ZOI分别在WT(A)和Δgt突变体(B)中分别为0.55(±0.1)cm和0.8(±0.08)cm。羧苄青霉素的ZOI对于WT(C)为1.6(±0.19)cm,对于Δdeg(D)为2.4(±0.25)cm。粘虫素类似地影响了WT(E)和Δdeg(F),ZOI为0.4厘米。黑色箭头表示测量的抑制区域。改编自Cheregi等人。 (2015)。


通过四个生物重复计算抑制面积(cm)和标准偏差的平均直径。每个生物复制品由3个技术重复表示;技术复制呈现相同的抑制区域。这些数据显示在Cheregi等人的表3和图3中。 (2015)。




  1. 微量矿物质(1升)
    2.86g H 3 3 3
    1.81g MnCl 2·4H 2 O 2/
    0.22g ZnSO 4·7H 2 O
    0.39g Na 2 MoO 4·2H 2 O
    0.079g CuSO 4·5H 2 O
    0.049g Co(NO 3 3)2·6H 2 O
  2. 100倍BG11无铁,磷酸盐,碳酸盐(1升)
    149.6g NaNO 3
    7.5g MgSO 4·7H 2 O·
    3.6g CaCl 2·2H 2 O ○
    1.12ml 0.25M Na 2 -EDTA,pH8.0
  3. 其他组件
    柠檬酸铁铵,6mg/ml(1,000x):600mg/100ml dH 2 O
    Na 2 CO 3(1,000×):2g Na 2 CO 3/100ml/100ml/2 O
    K 2 O 3 HPO 4(1,000×):3.05g K 2 HPO 4 /每100ml dH 3 2 O
  4. BG11液体介质(1升)

    1毫升1,000×K 2 HPO 4
  5. BG11固体琼脂平板(1升)
    10 ml 1 M TES/NaOH缓冲液pH 8.2(长期储存在冰箱中)
    15克Difco Bacto-agar
  6. 1 M TES/NaOH缓冲液,pH 8.2
    在蒸馏的H 2 O中溶解229.2g的TES 所得到的解决方案应该是清楚的


这项工作得到了瑞典能源机构(C. Funk)的资助。


  1. Bauer,A.W.,Kirby,W.M.M.,Sherris,J.C.和Turck,M。(1966)。 通过标准化单磁盘方法进行的抗生素敏感性测试。   Am J Clin Pathol 45(4):493-496。
  2. Cheregi,O.,Miranda,H.,Gröbner,G.and Funk,C。(2015)。 蓝藻中的Deg蛋白酶家族灭活集胞藻 sp。 PCC 6803对外部单元格层有影响。 < J Photoch Photobio B 152:383-394。
  3. Cheregi,O.,Wagner,R。和Funk,C。(2016)。 了解蓝细菌Deg/HtrA蛋白酶。  Front Plant Sci 7:624。
  4. Miranda,H.,Cheregi,O.,Netotea,S.,Hvidsten,T.R.,Moritz,T.and Funk,C。(2013)。 共表达分析,蛋白质组学和代谢组学研究的影响Deg/HtrA蛋白酶三重突变体在集胞藻中。 PCC 6803暴露于温度和高光照条件下。           78:294-311。
  5. Tam,L.X.,Aigner,H.,Timmerman,E.,Gevaert,K.and Funk,C。(2015)。 鉴定三种Deg/HtrA蛋白酶底物的蛋白质组学方法蓝细菌集胞藻PCC 6803。
    生物化学J 468(3):373-384。
  6. Trautner,C.和Vermaas,W. F.(2013)。 sll1951 基因编码表层集胞藻的蛋白质菌株PCC 6803。 < J Bacteriol 195(23):5370-5380。

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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Cheregi, O. and Funk, C. (2016). Antibiotic Disc Assay for Synechocystis sp. PCC6803. Bio-protocol 6(24): e2071. DOI: 10.21769/BioProtoc.2071.