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The presence of intracellular glycogen can be detected by the following iodine staining technique. Cells with glycogen stain dark brown, whereas in its absence they remain with a pale yellowish color. It is hypothesized that iodine atoms fit into helical coils formed by the α-polyglucan to form a coloured glycogen-iodine complex. Here, we have studied the expression of Streptococcus mutans (S. mutans) genes that control the biosynthesis of this polysaccharide (Asencion Diez et al., 2013). Thus, we expressed glgC and glgD genes coding for both ADP-Glc pyrophosphorylase subunits in Escherichia coli (E. coli) AC70R1-504 cells to complement the deficient accumulation of glycogen by this strain (Iglesias et al., 1993). In control cells or in those where an inactive protein was expressed, the synthesis of the polysaccharide was undetectable by this iodine staining technique.

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Iodine Staining of Escherichia coli Expressing Genes Involved in the Synthesis of Bacterial Glycogen
参与细菌糖原合成的大肠杆菌表达基因的碘染色

微生物学 > 微生物生物化学 > 糖类
作者: Ana M. Demonte
Ana M. DemonteAffiliation: Biochemistry and Biological Sciences, National University of the Litoral, Santa Fe, Argentina
Bio-protocol author page: a1619
Matías D. Asención Diez
Matías D. Asención DiezAffiliation: National University of the Litoral, National Council for Research in Science and Technology (CONICET), Santa Fe, Argentina
Bio-protocol author page: a1620
Sergio A. Guerrero
Sergio A. GuerreroAffiliation: Universidad Nacional del Litora, Argentina and Researcher of the National Council of Science (CONICET), Santa Fe, Argentina
Bio-protocol author page: a1621
Miguel A. Ballicora
Miguel A. BallicoraAffiliation: Department of Chemistry and Biochemistry, Loyola University, Chicago, USA
Bio-protocol author page: a1622
 and Alberto A. Iglesias
Alberto A. IglesiasAffiliation: Instituto de Agrobiotecnología del Litoral (UNL-CONICET), Facultad de Bioquímica y Ciencias Biológicas, Santa Fe, Argentina
For correspondence: iglesias@fbcb.unl.edu.ar
Bio-protocol author page: a1623
Vol 4, Iss 17, 9/5/2014, 3466 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1224

[Abstract] The presence of intracellular glycogen can be detected by the following iodine staining technique. Cells with glycogen stain dark brown, whereas in its absence they remain with a pale yellowish color. It is hypothesized that iodine atoms fit into helical coils formed by the α-polyglucan to form a coloured glycogen-iodine complex. Here, we have studied the expression of Streptococcus mutans (S. mutans) genes that control the biosynthesis of this polysaccharide (Asencion Diez et al., 2013). Thus, we expressed glgC and glgD genes coding for both ADP-Glc pyrophosphorylase subunits in Escherichia coli (E. coli) AC70R1-504 cells to complement the deficient accumulation of glycogen by this strain (Iglesias et al., 1993). In control cells or in those where an inactive protein was expressed, the synthesis of the polysaccharide was undetectable by this iodine staining technique.
Keywords: Reserve polysaccharide(储备多糖), Bacterial metabolism(细菌的代谢), Qualitative analysis(定性分析), ADP-glucose(ADP-葡萄糖), Glycogenesis(糖原的合成)

[Abstract] 细胞内糖原的存在可以通过以下碘染色技术检测。 具有糖原染色的细胞为深棕色,而在其不存在时,它们保持淡黄色。 假设碘原子适合由α-聚葡萄糖形成的螺旋线圈以形成有色的糖原 - 碘络合物。 在这里,我们研究了控制该多糖的生物合成的变异链球菌((变异链球菌))基因的表达(Asencion Diez等人 ,2013)。 因此,我们表达编码大肠杆菌中的ADP-Glc焦磷酸化酶亚基的 glgC 和 glgD 基因( >)AC70R1-504细胞以补充该菌株的糖原的不足积累(Iglesias等人,1993)。 在对照细胞或表达无活性蛋白质的那些细胞中,通过碘染色技术检测不到多糖的合成。

Materials and Reagents

  1. Cells: Non-transformed E. coli AC70R1-504 or harboring plasmids with the S. mutans glgC and glgD genes, separately or combined
    Note: This strain has a deficient production of the ADP-glucose pyrophosphorylase enzyme in absence of complementary plasmids (Morán-Zorzano et al., 2007).
  2. Luria-Bertani (LB) liquid medium
  3. Antibiotics: Kanamycin (US Biological, catalog number: K0010 ) and spectinomycin (Sigma-Aldrich, catalog number: S4014 )
  4. Inducers: Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich, catalog number: I6758 ) and nalidixic acid (Sigma-Aldrich, catalog number: N4382 )
  5. Plasmids: pMAB6/glgC, expressing S. mutans glgC (induced by nalidixic acid) and pMAB5/glgD, expressing S. mutans glgD (induced by IPTG)
    Note: These plasmids are compatible and bear resistance to spectinomycin and kanamycin, respectively (Asencion Diez et al., 2013).
  6. D(+) Glucose (Sigma-Aldrich, catalog number: G5767 )
  7. Iodine crystals (Biopack Medical, catalog number: 2000162300 )

Equipment

  1. 1.5 ml microcentrifuge tube (Deltalab, catalog number: 200400 )
  2. Microcentrifuge (Beckman Coulter, model: 22R )
  3. Shaker (at least 200 rpm) at 37 °C (Thermo Fisher Scientific)

Procedure

  1. Inoculate non-transformed and transformed E. coli AC70R1-504 cells onto 3 ml of LB medium with appropriate antibiotics to ensure plasmid permanence. Then, grow at 37 °C until an OD600 ~0.8 is reached. Usually the initial inoculate comes from a saturated culture.
    Induce protein expression for 3 h at 200 rpm and 25 °C in presence of IPTG, nalidixic acid or both, depending on the genes being expressed.
    Note: Temperature, inducer concentration and other conditions are specific for the system in order to assure a correct expression level of soluble and active protein.
  2. Afterwards, to stimulate glycogen production, add glucose to a final concentration of 0.2% (wt/vol) and further incubate (under the same conditions) for 1 h.
  3. Withdraw an aliquot of 0.1 ml and centrifuge it in a 1.5 ml microcentrifuge tube at 16,000 x g for 5 min. Aspirate supernatant carefully, leaving a compact cell pellet in the bottom of the tube.
    Note: The pellet should be as dried and compact as possible to avoid drops sliding down the walls of the tube.
  4. Turn upside down the microcentrifuge tube, so an iodine crystal could be placed in the cap of the closed tube. After 5 min at room temperature, iodine vapors stain the cell pellet if glycogen has been previously produced.
    Note: The specific amount of iodine in the crystal is not critical, as it only provides an excess of the compound present in vapors.

Representative data



Figure 1. Iodine staining of cells accumulating different amounts of glycogen. Iodine staining of cell pellets from E. coli AC70RI-504 (from left to right): Untransformed cells (lacking ADP-Glc PPase, negative control), transformed to express GlgC (catalytic subunit of ADP-Glc PPase forming homotetrameric enzyme, low positive sample), transformed to express GlgD (non-catalytic, inactive, subunit of ADP-Glc PPase, negative sample), or to express GlgC/GlgD (heterotetrameric ADP-Glc PPase, high positive sample). The staining is mainly qualitative and as much it could be established a semi-quantitative scale based on the visualization by eye (or scanner).

Notes

  1. The culture conditions and induction of recombinant protein synthesis (temperature, concentration of antibiotics and inducers) should be appropriate for other specific cases. If the growth of different cells varies, the procedure should be adjusted to make sure similar OD are reached to ensure similar metabolic conditions.
  2. After 5 min, remove iodine crystal to prevent darkening of the walls of the microtube to facilitate the acquisition of the image. The staining may fade always after few hours, but they could be re-stained.
  3. Before exposure to iodine vapors, the pellet should be as dried and compact as possible to avoid drops sliding down the walls of the tube, which will cause smearing. After aspiration, a small and long piece of paper filter could be used to remove tiny amounts of the remaining liquid.
  4. Traditionally, this type of staining has been done in plates (Greene et al., 1996). But, one of the advantages of this technique is that it could be used with any type of cells and expression systems, such as BL21 (DE3) E. coli and pET derivative vectors (Ballicora et al., 2007; Kuhn et al., 2010). In those cases, cells cannot be efficiently grown in plates in presence of IPTG.

Acknowledgments

This work was supported by grants to AAI from CONICET [PIP 2519 and CONICET-NSF 19537/28/06/12], UNL [CAI+D Orientado and Redes], and ANPCyT [PICT’08 1754]; and to MAB from the NSF [MCB 1024945]. MDAD is fellow from CONICET; SAG and AAI are investigators from the same institution.

References

  1. Asencion Diez, M. D., Demonte, A. M., Guerrero, S. A., Ballicora, M. A. and Iglesias, A. A. (2013). The ADP-glucose pyrophosphorylase from Streptococcus mutans provides evidence for the regulation of polysaccharide biosynthesis in Firmicutes. Mol Microbiol 90(5): 1011-1027.
  2. Ballicora, M. A., Erben, E. D., Yazaki, T., Bertolo, A. L., Demonte, A. M., Schmidt, J. R., Aleanzi, M., Bejar, C. M., Figueroa, C. M., Fusari, C. M., Iglesias, A. A. and Preiss, J. (2007). Identification of regions critically affecting kinetics and allosteric regulation of the Escherichia coli ADP-glucose pyrophosphorylase by modeling and pentapeptide-scanning mutagenesis. J Bacteriol 189(14): 5325-5333.
  3. Greene, T. W., Chantler, S. E., Kahn, M. L., Barry, G. F., Preiss, J. and Okita, T. W. (1996). Mutagenesis of the potato ADPglucose pyrophosphorylase and characterization of an allosteric mutant defective in 3-phosphoglycerate activation. Proc Natl Acad Sci U S A 93(4): 1509-1513.
  4. Iglesias, A. A., Barry, G. F., Meyer, C., Bloksberg, L., Nakata, P. A., Greene, T., Laughlin, M. J., Okita, T. W., Kishore, G. M. and Preiss, J. (1993). Expression of the potato tuber ADP-glucose pyrophosphorylase in Escherichia coli. J Biol Chem 268(2): 1081-1086.
  5. Kuhn, M. L., Figueroa, C. M., Aleanzi, M., Olsen, K. W., Iglesias, A. A. and Ballicora, M. A. (2010). Bi-national and interdisciplinary course in enzyme engineering. Biochem Mol Biol Educ 38(6): 370-379.
  6. Morán-Zorzano, M. T., Alonso-Casajús, N., Muñoz, F. J., Viale, A. M., Baroja-Fernández, E., Eydallin, G. and Pozueta-Romero, J. (2007). Occurrence of more than one important source of ADPglucose linked to glycogen biosynthesis in Escherichia coli and Salmonella. FEBS Lett 581(23): 4423-4429.

材料和试剂

  1. 细胞:未转化的。 大肠杆菌 AC70R1-504或含有质粒的质粒。 mutans glgC 和 glgD 基因,
    注意:该菌株在没有互补质粒的情况下缺乏ADP-葡萄糖焦磷酸化酶的产生(Morán-Zorzano等,2007)。
  2. Luria-Bertani(LB)液体培养基
  3. 抗生素:卡那霉素(US Biological,目录号:K0010)和壮观霉素(Sigma-Aldrich,目录号:S4014)
  4. 诱导剂:异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(Sigma-Aldrich,目录号:I6758)和萘啶酸(Sigma-Aldrich,目录号:N4382)
  5. 质粒:pMAB6/emgglC ,表达 S. mutans glgC (由萘啶酸诱导)和pMAB5/emggD ,表达。 mutan glgD (由IPTG诱导)
    注意:这些质粒是相容的,分别对壮观霉素和卡那霉素有抗性(Asencion Diez et al。,2013)。。
  6. D(+)葡萄糖(Sigma-Aldrich,目录号:G5767)
  7. 碘晶体(Biopack Medical,目录号:2000162300)

设备

  1. 1.5ml微量离心管(Deltalab,目录号:200400)
  2. 微量离心机(Beckman Coulter,型号:22R)
  3. 在37℃(Thermo Fisher Scientific)摇动(至少200rpm)

程序

  1. 接种未转化和转化的E.大肠杆菌AC70R1-504细胞接种到具有合适抗生素的3ml LB培养基上以确保质粒的持久性。然后,在37℃下生长直至达到OD 600〜0.8。通常初始接种来自饱和培养物 根据所表达的基因,在IPTG,萘啶酸或两者的存在下,在200rpm和25℃下诱导蛋白表达3小时。
    注意:温度,诱导剂浓度和其他条件是系统特异性的,以确保可溶性和活性蛋白的正确表达水平。
  2. 然后,为了刺激糖原生成,加入葡萄糖至终浓度为0.2%(wt/vol),并进一步孵育(在相同条件下)1小时。
  3. 取出0.1ml的等分试样,并将其在1.5ml微量离心管中以16,000×g离心5分钟。小心吸出上清液,在管底部留下紧密的细胞沉淀。
    注意:颗粒应尽可能干燥和紧凑,以避免液滴从管壁上滑落。
  4. 将微量离心管倒置,将碘晶体放入封闭管的盖子中。在室温下5分钟后,如果先前已经产生糖原,碘蒸气使细胞沉淀物染色。
    注意:晶体中碘的具体含量并不重要,因为它只能提供蒸气中存在的化合物过量。

代表数据



图1.积累不同量的糖原的细胞的碘染色。来自E的细胞沉淀的碘染色。大肠杆菌AC70RI-504(从左到右):转化以表达GlgC(形成同型四聚体酶的ADP-Glc PPase的催化亚基,低阳性样品)的未转化细胞(缺乏ADP-Glc PPase,阴性对照)以表达GlgD(非催化,非活性,ADP-Glc PPase的亚基,阴性样品)或表达GlgC/GlgD(异四聚体ADP-Glc PPase,高阳性样品)。染色主要是定性的,并且尽可能多地基于通过眼睛(或扫描仪)的可视化建立半定量量表。

笔记

  1. 培养条件和重组蛋白合成的诱导(温度,抗生素和诱导剂的浓度)应适用于其他特定情况。如果不同细胞的生长不同,应调整程序以确保达到类似的OD,以确保类似的代谢条件
  2. 5分钟后,取出碘晶体,以防止微管壁变黑,便于获取图像。染色可能会在几个小时后褪色,但它们可以重新染色
  3. 在暴露于碘蒸气之前,颗粒应当尽可能干燥和紧凑,以避免液滴从管壁滑落,这将导致涂抹。抽吸后,可以使用一个小而长的纸过滤器去除少量的剩余液体
  4. 传统上,这种类型的染色在板中进行(Greene等人,1996)。但是,该技术的优点之一是其可以用于任何类型的细胞和表达系统,例如BL21(DE3)E。大肠杆菌和pET衍生物载体(Ballicora等人,2007; Kuhn等人,2010)。在这些情况下,在IPTG存在下,细胞不能在平板中有效生长

致谢

这项工作得到了对来自CONICET的AAI的批准[PIP 2519和CONICET-NSF 19537/28/06/12],UNL [CAI + D Orientado和Redes]和ANPCyT [PICT'08 1754]的支持;和来自NSF的MAB [MCB 1024945]。 MDAD是CONICET的同胞; SAG和AAI是来自相同机构的研究者。

参考文献

  1. Asencion Diez,M.D.,Demonte,A.M.,Guerrero,S.A.,Ballicora,M.A。和Iglesias,A.A。(2013)。 来自链球菌突变体的ADP-葡萄糖焦磷酸化酶提供了调节在Firmicutes中的多糖生物合成。 Mol Microbiol 90(5):1011-1027。
  2. Ballicora,MA,Erben,ED,Yazaki,T.,Bertolo,AL,Demonte,AM,Schmidt,JR,Aleanzi,M.,Bejar,CM,Figueroa,CM,Fusari,CM,Iglesias,AAand Preiss, (2007)。 严重影响大肠杆菌ADP的动力学和变构调节的区域的鉴定葡萄糖焦磷酸化酶通过建模和五肽扫描诱变。细菌189(14):5325-5333。
  3. Greene,T.W.,Chantler,S.E.,Kahn,M.L.,Barry,G.F.,Preiss,J.and Okita,T.W。(1996)。 马铃薯ADP葡萄糖焦磷酸化酶的诱变和3-磷酸甘油酸激活中有缺陷的变构突变体的表征。 a> Proc Natl Acad Sci USA 93(4):1509-1513。
  4. Iglesias,A.A.,Barry,G.F.,Meyer,C.,Bloksberg,L.,Nakata,P.A.,Greene,T.,Laughlin,M.J.,Okita,T.W.,Kishore,G.M.and Preiss,J。(1993)。 在大肠杆菌中表达马铃薯块茎ADP-葡萄糖焦磷酸化酶。 J Biol Chem 268(2):1081-1086。
  5. Kuhn,M.L.,Figueroa,C.M.,Aleanzi,M.,Olsen,K.W.,Iglesias,A.A。和Ballicora,M.A。(2010)。 酶工程中的跨国和跨学科课程 生化分子生物学 38(6):370-379。
  6. Morán-Zorzano,M.T.,Alonso-Casajús,N.,Muñoz,F.J.,Viale,A.M.,Baroja-Fernández,E.,Eydallin,G.and Pozueta-Romero,J.(2007)。 与大肠杆菌中糖原生物合成相关的ADP葡萄糖的一个以上重要来源的发生 em>和沙门氏菌。 FEBS Lett 581(23):4423-4429。
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How to cite this protocol: Demonte, A. M., Diez, M. D., Guerrero, S. A., Ballicora, M. A. and Iglesias, A. A. (2014). Iodine Staining of Escherichia coli Expressing Genes Involved in the Synthesis of Bacterial Glycogen. Bio-protocol 4(17): e1224. DOI: 10.21769/BioProtoc.1224; Full Text



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