Rapid Preparation of Unsheathed Bacterial Flagella

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Molecular Microbiology
Dec 2013



The flagellum is required for bacterial swimming and swarming motility. In the biphasic Salmonella enterica serovar Typhimurium (S. Typhimurium), the flagellar filament is build up by two distinct monomeric subunits, flagellin FliC and FljB. S. Typhimurium has the ability to switch between two flagellins, FliC and FljB, in a phase-variable manner. The switch to FliC is called phase H1 and considered important for bacterial growth and survival in the spleen in a murine infection model of typhoid fever. Flagellin is secreted as monomeric subunits, but the majority of flagellin is polymerized upon secretion as the flagellar filament. Salmonella flagellin has traditionally been isolated through a process involving multiple steps of centrifugation and acid treatment. Here, we delineate a simplified protocol for preparing Salmonella´s flagellin for analytical purpose to determine the amount of flagellin without the aid of antibodies. The growth conditions used were stationary phase, logarithmic phase and a low oxygen and high salt condition mimicking the gastrointestinal tract. Flagellin expression of other source organisms, such as other serovars of Salmonella enterica and Escherichia coli, including flagellar phase- or genetic variants can be analysed. Flagellin expression analysis complements flagella-associated phenotype analysis such as swimming and swarming behaviour.

Keywords: Bacteria (细菌), Salmonella (沙门氏菌), Flagellin (鞭毛蛋白), Flagellar phase variation (鞭毛的相位变化)

Materials and Reagents

  1. Bacterial strains (S. Typhimurium)
  2. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  3. 100% trichloroacetic acid (Sigma-Aldrich, catalog number: T6399 )
  4. β-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  5. Trizma® base (powder) (Sigma-Aldrich, catalog number: T1503 )
  6. 1 M HCl solution (Sigma-Aldrich, catalog number: H1758 )
  7. Glycerol 1 L (Sigma-Aldrich, catalog number: 49781 )
  8. Sodium dodecyl sulfate - 500 g (Sigma, catalog number: L4390 )
  9. Tryptone (BD-catalog number: 211705 )
  10. Yeast extract (BD, catalog number: 212750 )
  11. Agar (BD, catalog number: 281230 )
  12. Bromophenol blue (Shelton scientific, catalog number: IB74040 )
  13. Glycerol stock for one vial (see Recipes)
  14. LB medium (see Recipes)
  15. 1 M Tris (see Recipes)
  16. 2x SDS sample buffer (see Recipes)


  1. Falcon tubes (50 ml and 15 ml) (TPP, catalog numbers: 91050 and 91115 )
  2. Filter Flasks (pore size 0.22 μm diameter) (TPP, catalog number: 99500 )
  3. Eppendorf tubes (1.5 ml) (Sarstedt, catalog number: 72.690.001 )
  4. Incubator (adjusted to 37 °C) (Memmert, model: ULE500 )
  5. Rotary shaker (HTINFORS, model: Minitron )
  6. Spectrophotometer (Hitachi, model: U1100 )
  7. Thermoblock (PEQLAB, model: 1202 )
  8. Vortex mixer (VWR International, model: 1719 )
  9. Luer lock tips (BD PLASTIKPAK, catalog number: 302187 )
  10. Needle (0.51 mm outer diameter) (BD Microlance sterile, model25 g x 5/8” x 100)
  11. Sterile petri dishes (90 mm) (Sartorius, catalog number: 14-555-735 )
  12. Centrifuge (HERAEUS, model: PICO17 )
  13. Microcentrifuge (NeoLab, model: microcentrifuge II )
  14. pH meter (Hanna instruments, model: pH211 )


  1. Preparation of the pre-culture (to be used for all subsequent culture conditions)
    Day 0
    Streak bacterial strains from -80 °C glycerol stock [equal to 87% (v/v) glycerol in LB] on LB agar plates for single colonies. Use LB agar plates containing appropriates antibiotics, if required, and incubate overnight at 37 °C (approximately 16 to 24 h).
    Day 1
    1. Take one bacterial colony and inoculate in 10 ml liquid LB medium (in a 50 ml Falcon tube).
    2. Incubate the culture overnight at 37 °C with shaking at 150 rpm. Put the lid loose on the tube. Add appropriate antibiotics, if required.

  2. Preparation of inoculum
    Day 2
    1. Measure the optical density of the 1:10 diluted pre-culture in a spectrophotometer and calculate the optical density of the pre-culture. Adjust the OD600 of the pre-culture to OD600=1 for a 1 ml volume which corresponds to approximately 5 x 108 CFU.
    2. Add 100 μl of the adjusted pre-culture to 10 ml LB medium (1:100 dilution).

  3. Culture conditions

    Standard growth condition to logarithmic and stationary phase in LB medium
    Add 100 µl of wild type, normalized test and control inoculum to 10 ml LB medium in a 50 ml Falcon tube (a 1:100 dilution of the inoculum). Add appropriate antibiotics and inducer if required.
    1. Logarithmic phase growth condition
      1. Incubate the samples for 3 h at 37 °C with shaking at 150 rpm. Put the lid loose on the tube. The OD600 will be approximately 0.6-0.8.
      2. Normalize each sample to OD600 = 0.6. Shear off flagella as described under D.
    2. Stationary phase growth condition
      1. Incubate the samples for 16 to 24 h at 37 °C with shaking at 150 rpm. Put the lid loose on the tube.
      2. Normalize each sample to OD600 = 0.6. Shear off flagella as described under D.

    S. Typhimurium cell culture invasion condition
    1. Low oxygen, high salt growth condition.
      1. Add 100 µl wild type, normalized test and control inoculum to 10 ml LB medium with high salt concentration in a 50 ml Falcon tube (a 1:100 dilution of the inoculum). Add appropriate antibiotics and inducer, if required.
      2. Incubate the samples at 37 °C with shaking at 150 rpm until OD600 = 0.6-0.7 (takes approximately 4.5 h). Put the lid loose on the tube.
      3. Normalize the OD600 of the samples to the wild type OD600 using LB medium with high salt concentration. Shear off flagella as described under D.

  4. Shearing off cell-associated flagellin (flagella)
    1. Perform the following steps on ice.
    2. Place 5 ml of each normalized sample in an individual sterile petri dish plate.
    3. Lift the inoculum up from the sterile petri dish plate with a sterile Luer lock tip (syringe). Place the sterile needle on the Luer lock tip and eject the cell suspension back in the petri dish plate. Work on a clean bench as this procedure creates aerosol.
    4. Repeat the previous step 10 times with the same material to shear off cell-associated flagella.
    5. Take 1 ml of cell suspension and transfer to an Eppendorf tube. Centrifuge at 17,700 x g for 10 min at 4 °C.
    6. Transfer 800 µl of the supernatant to a new Eppendorf tube. The supernatant contains sheared-off flagella and minor amounts of secreted flagellin. Proceed with E, for flagellar preparation.
    7. Remove the rest of the supernatant. The bacterial pellet is used for analysis of cell-associated protein as described under E, for cell associated protein preparation.

  5. Sample preparation

    For flagellar preparation
    1. Centrifuge at 17,700 x g for 10 min at 4 °C.
    2. Transfer 750 µl to a new Eppendorf tube.
    3. Add 250 μl of cold trichloroacetic acid solution and mix gently.
    4. Place the sample for at least 1 h on ice or in a -20 °C freezer.
    5. Centrifuge at 17,700 x g for 40 min at 4 °C, remove the supernatant and remove the residual liquid (eventually through repeated centrifugation).

    For cell associated protein preparation
    1. Centrifuge 17,700 x g for 1 min at 4 °C.
    2. Discard the rest of the supernatant and save the bacterial pellet.

    Preparation of cell associated protein and flagellin samples for loading on protein gel
    1. Prepare the sample buffer with 90 (v/v)% 1x SDS sample pre-buffer and 10 (v/v)% β-mercaptoethanol.
    2. Resuspend the flagellar protein preparation in 45 μl sample buffer and 5 μl Tris-HCl (pH = 9).
    3. Resuspend the bacterial pellet in 100 μl sample buffer.
    4. Vortex the samples.
    5. Heat up the samples 5 min at 95 °C.
    6. Vortex the samples.
    7. Spin down with a microcentrifuge for few seconds.
      The samples can be used immediately or stored at -20 °C for further utilization.

Representative data

Figure 1. Coomassie brilliant blue stained 12% SDS PAGE of flagellar preparations from Salmonella enterica serovar Typhimurium UMR1 wild type and isogenic flagellin mutants. M. PageRuler prestained protein ladder 1. Wild type: ATCC14028-1s Nalr; 2. UMR1 fliC5050::MudJ fljB5001::MudCmr; 3. UMR1 fljB5001::MudCmr; 4. UMR1 fliC5050::MudJ. Molecular weight of flagellin: FliC: 52 kDa, FljB: 53 kDa


  1. Bacterial strains and controls.
    This protocol has been developed using the wild type strain S. enterica serovar Typhimurium UMR1 (ATCC14028-1s Nalr). In this case, control strains are isogenic fljB fliC, fljB and fliC mutants. These strains are available upon request from the corresponding author.
  2. Medium volumes and culture conditions.
    These factors can be subjected to change. Growth medium, medium volume and time of incubation can be adapted to the research question.
  3. Samples are normalized to OD600 = 1 in step C1b, 2b and 3c in order to enable growth culture independent estimation of flagellin expression.
  4. The cell associated protein samples.
    The cell associated protein samples will be used to assess whether equal amounts of cells have been used for flagellar preparation.
  5. Precipitation of flagellar preparation samples.
    The samples can be stored at -20 °C after adding trichloroacetic acid solution to the flagellin sample.
  6. A supplementary centrifugation of 5 min at 17,700 x g between step C and D can be used to separate polymerized and monomeric secreted flagellin.


  1. Glycerol stock for one vial
    250 μl 87% glycerol
    750 μl bacterial overnight culture
  2. LB medium (1 L)
    10 g tryptone
    5 g yeast extract
    10 g NaCl
    Variant: with high salt concentration 17.5 g NaCl (0.3 M NaCl)
    Fill up to 1 L with demineralized water and adjust the medium to pH = 7
    Sterilize by autoclaving or filtration
  3. 1 M Tris (adjusted to pH = 9)
    Add 12.14 g of Trizma® to deionized water for a total volume of 100 ml
    Adjust the pH to 9 with 1 M HCl solution
  4. 2x SDS sample buffer
    25 ml 0.5 M Trizma-base (pH = 6.8)
    20 ml glycerol 87%
    4 g SDS
    0.2 g Bromophenol-blue
    45 ml H2O
    Before utilization, add 10% β-mercaptoethanol to 90% of 1x sample buffer


The development of this protocol was supported by research grants from Karolinska Institutet Doctoral funding (project no. C112130052) and the Swedish Research Council Natural Sciences and Engineering (project no. 621-2010-5755). The authors have no conflict of interest to declare. The protocol described above is adapted from one reported previously (Le Guyon et al., 2014; Ahmad et al., 2013). Kelly T. Hughes kindly provided fliC5050::MudJ and fljB5001::MudCmr mutant alleles in S. Typhimurium LT2.


  1. Ahmad, I., Wigren, E., Le Guyon, S., Vekkeli, S., Blanka, A., El Mouali, Y., Anwar, N., Chuah, M. L., Lünsdorf, H., Frank, R., Rhen, M., Liang, Z. X., Lindqvist, Y. and Römling, U. (2013). The EAL-like protein STM1697 regulates virulence phenotypes, motility and biofilm formation in Salmonella typhimurium. Mol Microbiol 90(6): 1216-1232.
  2. Ikeda, J. S., Schmitt, C. K., Darnell, S. C., Watson, P. R., Bispham, J., Wallis, T. S., Weinstein, D. L., Metcalf, E. S., Adams, P., O'Connor, C. D. and O'Brien, A. D. (2001). Flagellar phase variation of Salmonella enterica serovar Typhimurium contributes to virulence in the murine typhoid infection model but does not influence Salmonella-induced enteropathogenesis. Infect Immun 69(5): 3021-3030.
  3. Ibrahim, G. F., Fleet, G. H., Lyons, M. J. and Walker, R. A. (1985). Method for the isolation of highly purified Salmonella flagellins. J Clin Microbiol 22(6): 1040-1044.
  4. Guyon, S. L., Simm, R., Rehn, M. and Romling, U. (2014). Dissecting the cyclic di-guanylate monophosphate signalling network regulating motility in Salmonella enterica serovar Typhimurium. Environ Microbiol. (Epub ahead of print)
  5. Rochon, M. and Römling, U. (2006). Flagellin in combination with curli fimbriae elicits an immune response in the gastrointestinal epithelial cell line HT-29. Microbes Infect 8(8): 2027-2033.
  6. Römling, U. and Rohde, M. (1999). Flagella modulate the multicellular behavior of Salmonella typhimurium on the community level. FEMS Microbiol Lett 180(1): 91-102.
  7. Römling, U., Bian, Z., Hammar, M., Sierralta, W. D. and Normark, S. (1998). Curli fibers are highly conserved between Salmonella typhimurium and Escherichia coli with respect to operon structure and regulation. J Bacteriol 180(3): 722-731.


鞭毛是细菌游泳和群居运动所需的。在双相沙门氏菌鼠伤寒沙门氏菌(Typhimurium)(Typhimurium)中,鞭毛丝由两种不同的单体亚单位鞭毛蛋白FliC和FljB组成。 Typhimurium具有以相变方式在两个鞭毛蛋白FliC和FljB之间切换的能力。切换到FliC被称为H1期,并且被认为对于鼠伤寒热的鼠感染模型中的脾的细菌生长和存活是重要的。鞭毛蛋白作为单体亚基分泌,但是大多数鞭毛蛋白在分泌时作为鞭毛丝聚合。沙门氏菌鞭毛蛋白通常通过包括多步离心和酸处理的方法分离。在这里,我们描述了用于制备沙门氏菌鞭毛蛋白的简化方案以用于分析目的,以在没有抗体的帮助下确定鞭毛蛋白的量。所用的生长条件是固定相,对数期和模拟胃肠道的低氧和高盐条件。可以分析其他来源生物体的鞭毛蛋白表达,例如沙门氏菌和大肠杆菌的其他血清型,包括鞭毛相或遗传变异体。鞭毛蛋白表达分析补充鞭毛相关表型分析,如游泳和蜂群行为。

关键字:细菌, 沙门氏菌, 鞭毛蛋白, 鞭毛的相位变化


  1. 细菌菌株( Typhimurium)
  2. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  3. 100%三氯乙酸(Sigma-Aldrich,目录号:T6399)
  4. β-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  5. Trizma底物(粉末)(Sigma-Aldrich,目录号:T1503)
  6. 1M HCl溶液(Sigma-Aldrich,目录号:H1758)
  7. 甘油1L(Sigma-Aldrich,目录号:49781)
  8. 十二烷基硫酸钠500g(Sigma,目录号:L4390)
  9. 胰蛋白胨(BD目录号:211705)
  10. 酵母提取物(BD,目录号:212750)
  11. 琼脂(BD,目录号:281230)
  12. 溴酚蓝(Shelton scientific,目录号:IB74040)
  13. 一个小瓶的甘油储备(见配方)
  14. LB介质(见配方)
  15. 1 M Tris(见配方)
  16. 2x SDS样品缓冲液(见配方)


  1. Falcon管(50ml和15ml)(TPP,目录号:91050和91115)
  2. 过滤瓶(孔径0.22μm直径)(TPP,目录号:99500)
  3. Eppendorf管(1.5ml)(Sarstedt,目录号:72.690.001)
  4. 孵育器(调节至37℃)(Memmert,型号:ULE500)
  5. 旋转振荡器(HTINFORS,型号:Minitron)
  6. 分光光度计(日立,型号:U1100)
  7. 热块(PEQLAB,型号:1202)
  8. 涡旋混合器(VWR International,型号:1719)
  9. Luer锁尖(BD PLASTIKPAK,目录号:302187)
  10. 针(0.51mm外径)(BD Microlance无菌,型号25g×5/8"×100)
  11. 无菌培养皿(90mm)(Sartorius,目录号:14-555-735)
  12. 离心机(HERAEUS,型号:PICO17)
  13. 微量离心机(NeoLab,型号:微量离心机II)
  14. pH计(Hanna instruments,型号:pH211)


  1. 预培养物的制备(用于所有后续培养条件)
    条纹细菌菌株从LB琼脂板上的-80℃甘油储液[等于在LB中的87%(v/v)甘油],用于单菌落。 如果需要,使用含有适当抗生素的LB琼脂平板,并在37℃(约16至24小时)孵育过夜。
    1. 取一个菌落并接种在10ml液体LB培养基(在50ml Falcon管中)
    2. 在37℃下以150rpm振荡培养过夜。 放   盖子松动在管上。 如果需要,加入适当的抗生素。

  2. 接种物的制备
    1. 测量1:10稀释的预培养物在a中的光密度 分光光度计并计算预培养物的光密度。 对于1ml体积,将预培养物的OD 600调节至OD 600 = 1。 对应于大约5×10 8 CFU
    2. 加入100μl的调整预培养至10ml LB培养基(1:100稀释)。

  3. 文化条件

    向50ml Falcon管(接种物的1:100稀释液)中的10ml LB培养基中加入100μl野生型,标准化的测试和对照接种物。 如果需要,添加适当的抗生素和诱导剂
    1. 对数期生长条件
      1. 孵育样品3小时 在37℃下以150rpm振荡。 将盖子松动放在管上。 的 OD 600 约为0.6-0.8
      2. 将每个样品标准化至OD 600 = 0.6。 如D所述剪切鞭毛。
    2. 静止期生长条件
      1. 孵育样品16至24小时,在37°C,在150 rpm的摇动。 将盖子轻轻放在试管上。
      2. 将每个样品标准化至OD 600 = 0.6。 如D所述剪切鞭毛。

    S。 鼠伤寒沙门氏菌细胞培养物侵袭条件
    1. 低氧,高盐生长条件。
      1. 加入100μl野生型, 标准化的测试和对照接种到具有高盐的10ml LB培养基中 在50ml Falcon管(接种物的1:100稀释液)中的浓度。   如果需要,添加适当的抗生素和诱导剂
      2. 孵化 样品在37℃下以150rpm振摇直至OD 600 = 0.6-0.7 (约需4.5小时)。 将盖子轻轻放在试管上。
      3. 使用LB将样品的OD 600标准化为野生型OD 600 介质具有高盐浓度。 如所述剪切鞭毛 下D.

  4. 剪切细胞相关鞭毛蛋白(鞭毛)
    1. 在冰上执行以下步骤。
    2. 将5ml的每个标准化样品置于单个无菌培养皿板中
    3. 从无菌培养皿板上提起接种物 无菌Luer锁头(注射器)。 将无菌针放在鲁尔 锁定尖端并将细胞悬浮液弹回培养皿盘中。 在干净的工作台上工作,因为此过程会产生气溶胶
    4. 用相同的材料重复前面的步骤10次,以剪除细胞相关的鞭毛
    5. 取1毫升细胞悬液,并转移到Eppendorf管。 在4℃下以17,700×g离心10分钟。
    6. 转移800微升的上清液到一个新的Eppendorf管。 的 上清液含有剪切的鞭毛和少量分泌的 鞭毛蛋白。 继续E,用于鞭毛准备。
    7. 去除 剩余的上清液。 细菌沉淀用于分析 细胞相关蛋白,如E所述,用于细胞相关 蛋白质制备。

  5. 样品准备

    1. 在4℃下以17,700×g离心10分钟。
    2. 转移750微升到一个新的Eppendorf管
    3. 加入250μl冷三氯乙酸溶液,轻轻混匀
    4. 将样品在冰上或-20°C冰箱中放置至少1小时
    5. 在4,700×g离心40分钟,除去上清液 并去除残留的液体(最终通过重复 离心)。

    1. 在4℃下离心17,700×g,历时1分钟。
    2. 丢弃剩余的上清液并保存细菌沉淀。

    1. 用90(v/v)%1x SDS样品预缓冲液和10(v/v)%β-巯基乙醇制备样品缓冲液。
    2. 将鞭毛蛋白制剂重悬在45μl样品缓冲液和5μlTris-HCl(pH = 9)中。
    3. 将细菌沉淀重悬于100μl样品缓冲液中
    4. 旋转样本。
    5. 在95℃下加热样品5分钟
    6. 旋转样本。
    7. 用微量离心机旋转几秒钟。


图1.考马斯亮蓝染色的12%SDS来自沙门氏菌沙门氏菌沙门氏菌鼠伤寒沙门氏菌UMR1野生型和同基因鞭毛蛋白突变体的鞭毛制备物。 M. PageRuler预染色的蛋白梯1.野生 类型:ATCC14028-1s Nal r ; 2. UMR1 fli 5050 :: mu d J flj :Mu Cm r ; 3. UMR1 flj B 5001 :: Mu Cm r ; 4. UMR1 fli C 5050 :: Mu 鞭毛蛋白的分子量:FliC:52kDa,FljB:53kDa


  1. 菌株和对照。
    该方案是使用野生型菌株开发的。肠毒性鼠伤寒沙门氏菌UMR1(ATCC14028-1s Nal r )。在这种情况下,对照菌株是同基因的 em> B 和 fli 这些菌株可根据相应作者的要求获得。
  2. 中等体积和培养条件。
  3. 在步骤C1b,2b和3c中将样品标准化为OD 600 = 1,以便能够独立于生长培养物评估鞭毛蛋白表达。
  4. 细胞相关蛋白样品 细胞相关蛋白样品将用于评估等量的细胞是否已用于鞭毛制备
  5. 鞭毛制剂样品的沉淀。
  6. 在步骤C和D之间以17,700×g进行5分钟的补充离心可用于分离聚合的和单体分泌的鞭毛蛋白。


  1. 一个小瓶的甘油储备
    250微升87%甘油 750微升细菌过夜培养物
  2. LB培养基(1L)
    变体:具有高盐浓度17.5g NaCl(0.3M NaCl)
    用去离子水填充至1 L,并将介质调节至pH = 7
  3. 1mM Tris(调节至pH = 9)
    向去离子水中加入12.14g Trizma ,总体积为100ml
    用1M HCl溶液将pH调节至9
  4. 2×SDS样品缓冲液
    25ml 0.5M Trizma碱(pH = 6.8) 20ml甘油87%
    4 g SDS
    45ml H 2 O 2 / 使用前,向90%的1x样品缓冲液中加入10%β-巯基乙醇


该协议的制定由Karolinska机构博士资助(项目编号C112130052)和瑞典研究委员会自然科学和工程(项目编号621-2010-5755)的研究拨款支持。作者没有声明利益冲突。上述方案改变自之前报道的方案(Le Guyon等人,2014; Ahmad等人,2013)。 Kelly T. Hughes热情地提供了 fli 5050 :: Mu J和 flj em> 5001 :: Mu Cmr突变体等位基因在鼠伤寒沙门氏菌LT2中的表达。


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引用:Guyon, S. L., Rhen, M. and Römling, U. (2015). Rapid Preparation of Unsheathed Bacterial Flagella . Bio-protocol 5(6): e1425. DOI: 10.21769/BioProtoc.1425.