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Transformation of Thermus Species by Natural Competence
通过自然竞争法转化栖热菌属细胞   

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Abstract

Many Thermus species harbour genomes scourged with horizontally transferred signatures. Thermus thermophilus (Tth) has been characterized as naturally competent. The transformation protocol described here is based on the maximum DNA uptake rates registered at mid-exponential phase (OD600 0.3-0.4). Here we describe the stepwise protocol followed for transformation of both plasmids and linearized genomic DNA, of which the latter can be employed as an alternative method to electroporation to introduce mutations or to generate gene deletions in Thermus isolates, for instance.

Background

Thermus thermophilus (Tth) is an extreme thermophilic species extensively used as laboratory model, due to its ancient phylogenetic origin, the comparative ease of crystallisation of its proteins and macromolecular complexes, and the fast growth and good yields under laboratory growth conditions of several of its isolates. Among the most commonly employed isolates, the strains T. thermophilus HB27 and HB8 constitute the most frequently used models due to the high rates by which they can be transformed by natural competence. DNA of any source and topology can be easily taken up by growing cells of these isolates at rates of around 40 kb/s/cell (Schwarzenlander and Averhoff, 2006) showing yields of around 10-2 transformants/viable cell. For this, a polar located natural competence apparatus (Gold et al., 2015) involving at least 16 proteins encoded in five loci in the chromosome (Averhoff, 2009) of both strains is expressed essentially in a constitutive way, although the transformation efficiency is higher at exponential phase. Variants of antibiotic resistance genes encoding thermostable variants have been developed by directed evolution as gene markers, in such a way that selection can be performed in plates with kanamycin (Lasa et al., 1992), hygromycin B (Nakamura et al., 2005), or bleomycin (Brouns et al., 2005). Streptomycin resistance can be also employed to check natural competence with isogenic DNA (Koyama et al., 1986). This protocol describes the highly efficient transformation of cultures at exponential growth phase, providing reproducible data of maximized transformation efficiency.

Materials and Reagents

  1. 12 ml sterile plastic tubes
  2. 15 ml sterile plastic tubes
  3. Sterile plastic Petri dish plates (standard size; 100 x15 mm)
  4. Sterile tips for micropippettes, 5-200 µl (for instance, Daslab, catalog number: 162001 )
  5. Sterile tips for micropippettes, 100-1,000 µl (for instance, Daslab, catalog number: 162222 )
  6. Sterile tips for micropippettes, 10 µl (for instance, Metler Toledo, catalog number: 17004280 )
  7. Recipient Thermus strain, for instance, HB27 wild type (HB27/ATCC BAA-163 /DSM 7039)#
    #Note: Other Thermus thermophilus strains such as NAR1 and HB8, have been defined as naturally competent, thus, harboring the competence proteins required for transformation (César et al., 2011). All Thermus spp. strains referred here are available at the DSMZ collection.
  8. Kanamycin sulphate (Sigma-Aldrich, catalog number: K1377-5G )
  9. Tryptone (Conda, catalog number: 1612 )
  10. Yeast extract (Conda, catalog number: 1702 )
  11. Sodium chloride (NaCl) (EMD Millipore, catalog number: 1.06404.1000 )
  12. Agar (grade A) (Conda, catalog number: 1800 )
  13. Thermus water (carbonated-rich mineral water)
  14. DNA template: purified plasmids or genomic DNA** (harbouring the thermostable kat cassette)
    **Note: DNA quantity depends on the topology of the DNA to be transformed and the competence of the recipient strain.
  15. TB liquid medium (see Recipes)
  16. TB agar plates (see Recipes)

Equipment

  1. 50 ml Erlenmeyer flask
  2. Rotary shaker incubator reaching 70 °C (150 rpm) (Thermo Scientific, Thermo ScientificTM, catalog number: SHKE 420HP )
  3. Glass beads or a spreader
  4. Autoclave device (CertoClav, model: EL 18L )
  5. Spectrophotometer (Hitachi, model: U-2000 )
  6. Pipette P2, 0.2-2 μl (Gilson, Pipetman classicTM, catalog number: F144801 )
  7. Pipette P20, 2-20 μl (Gilson, Pipetman classicTM, catalog number: F123600 )
  8. Pipette P100, 20-100 μl (Gilson, Pipetman classicTM, catalog number: F123615 )
  9. Pipette P200, 50-200 μl (Gilson, Pipetman classicTM, catalog number: F123601 )
  10. 70 °C heater (Gemini, model: Memmert UE400 )
  11. Humid chambers
    Note: Tupperware devices are preferentially used. Humidified plastic bags are also valid. Addition of a paper towel, soaked on Thermus water and laid on the bottom of the chamber would enhance humidity maintenance. Ventilation should be avoided in order to conserve humidity. An example is provided in the photographs beneath (Figure 1).


    Figure 1. Humid chambers. Descriptive photographs of the humid chambers used to incubate Thermus plates. Place a piece of paper soaked with Thermus water and a bottle cap filled with the same water on the bottom of the chambers to maintain high humidity.

Procedure

  1. Culture preparation: growth of the subject strain to be transformed (see Figure 2).
    1. Inoculate 50 μl of saturated liquid growth culture (optical density at 600 nm [OD600] aprox. 2) in 10 ml TB medium in a 50-ml Erlenmeyer or inoculate the culture from the glycerol stock or from a frozen pellet. In the latter cases, one additional day growth would be required to reach optimal culture fitness.
    2. Incubate overnight the culture at 65 °C in a shaker incubator at 150 rpm.
    3. Dilute the overgrown culture in 10 ml fresh TB medium to OD600 = 0.05 in a 50-ml Erlenmeyer.
      Notes:
      1. If the culture has not reached saturation after overnight growth, postposition of the experiment is highly recommended.
      2. If the culture has not grown optimally overnight and the experiment should be performed, a starting OD600 of 0.07-0.08 is possible.
    4. Incubate in a shaker incubator at 65 °C until an OD600 of 0.3-0.4 is reached.
      Note: An OD600 close to 0.3 is recommended.

      Figure 2. Schematic representation of the Thermus transformation procedure. Plate shows a dilution of a transformation of 100 ng of a linearized plasmid harbouring a kat cassette flanked by two genomic arms for double recombination in HB27 strain.

  2. DNA addition
    1. Add 0.01-1 μg of DNA (genomic ~0.01 μg; plasmids ~0.1-0.25 μg; PCR products ~1 μg) in a sterile 12 ml plastic tube. Tubes can be pre-heated at 65 °C.
      Notes:
      1. Genomic DNA (carrying a selectable marker integrated in the genome) should not be added in higher quantities to avoid recombination farther than the target.
      2. DNA samples should be purified following standard protocols and eluted in water or TE.
      3. Pre-heating of the tubes keeps the temperature of the growing culture stable.
    2. Add 500 μl of the exponentially growing culture (see Figure 2) to the DNA.
    3. As a control, prepare 15 ml plastic tube without DNA, and add 500 μl of the exponentially growing culture to this tube.
  3. DNA transformation
    1. Incubate each tube in a shaker incubator at 65 °C for 4 h.
    2. Plate 50 μl and 400 μl of the cells on selective agar medium, spread by using glass beads or a spreader.
      Notes:
      1. Volume plated depends on the competence efficiency of the employed Thermus strain. Previous assays plating serial dilution spots can shed light on the appropriate volume to plate.
      2. Plate the control tube like the others, in addition to plating on a non-selective agar medium, as it will work as the reference of viable cells.
    3. Incubate plates in humid chambers for 48 h at 65 °C or until colonies become visible.
      Notes:
      1. Ordinarily, incubation temperature is set according to optimal growth of the strain but it should be adapted to the limitations of the selectable marker employed in the transformations too (for instance, hph5 conferring hygromycin resistance cannot be exposed to temperatures higher than 60 °C whereas kanamycin is selectable in temperatures higher than 70 °C).
      2. Transformation yields highly depend on the DNA template employed and the strain being transformed. For instance, HB27 transformed with 100 ng of plasmidic DNA shows rates of 10-3 transformants per viable cell.

Data analysis

  1. For transformation assays, a set of statistical tests should be performed to compare and analyse the transfer frequencies obtained.
  2. Transfer frequencies are calculated as the number of CFU transformants divided by the number of CFU of viable cells. To examine differences among transfer frequencies, Student’s t-test and Mann-Whitney U-tests may be used. When comparing transformability of a strain testing several DNA templates, examination of the differences among and within various DNA templates transfer frequencies can be addressed by one-way ANOVA. If different strains are tested, Wilcoxon-tests may be used to evaluate differences among their transfer frequencies. Simple linear regression test can be implemented for modelling the relationship between the DNA template and the frequency of transfer. Post-hoc Tukey and Bonferroni tests should be applied when convenient. 
  3. At least, 3 independent experiments, should be performed for reliable and reproducible data.

Notes

  1. This transformation protocol was optimized for Thermus thermophilus HB27, which shows the highest natural competence efficiency within the genus and the highest DNA uptake rates. Other strains with less competence capability should be treated with larger quantities of DNA and/or longer incubation periods.
  2. In case transformation has been performed to introduce a mutation, transformants should be re-streaked on selective plates and further grown in liquid under each particular condition to ensure a stable mutation.
  3. DNA amount required varies from 10 ng when employing genomic DNA to 1,000 ng when employing PCR products. On average, replicative plasmids are employed at 100-250 ng.
  4. Regardless the selective marker employed, post-incubation times are at least 3 to 4 h at 65 °C and 150 rpm. If DNA is added at the start of the exponential phase (OD600 = 0.2), times delays up to 5 h incubation.

Recipes

  1. TB liquid medium
    0.8 % tryptone (w/v)
    0.4 % yeast extract (w/v)
    50 mM NaCl, pH 7.5
    Thermus water (carbonated-rich mineral water)*
    pH may be adjusted by the addition of NaOH
  2. TB agar plates
    0.8% tryptone (w/v)
    0.4% yeast extract (w/v)
    50 mM NaCl, pH 7.5
    1.8% agar (grade A) (w/v)
    Thermus water (carbonated-rich mineral water)*

*Note: The analytical composition of the spring mineral water employed, per mg/ml, is listed beneath (Table 1). Other mineral waters easily purchasable such as Evian water or a laboratory solution resembling this recipe, can be employed.

Table 1. Mineral composition of the spring water from Jaraba Spring in Zaragoza (Spain)

Acknowledgments

This work has been funded by Grants BIO2013-44963-R, and FP7-PEOPLE-2012-IAPP No. 324439. A. Blesa held an FPI contract from the Spanish Ministry of Economy and Competitivity.

References

  1. Averhoff, B. (2009). Shuffling genes around in hot environments: the unique DNA transporter of Thermus thermophilus. FEMS Microbiol Rev 33(3): 611-626.
  2. Brouns, S. J., Wu, H., Akerboom, J., Turnbull, A. P., de Vos, W. M. and van der Oost, J. (2005). Engineering a selectable marker for hyperthermophiles. J Biol Chem 280(12): 11422-11431.
  3. César, C. E., Alvarez, L., Bricio, C., van Heerden, E., Littauer, D. and Berenguer, J. (2011). Unconventional lateral gene transfer in extreme thermophilic bacteria. Int Microbiol 14(4): 187-199.
  4. Gold, V. A., Salzer, R.; Averhoff, B. and Kühlbrandt, W. (2015). Structure of a type IV pilus machinery in the open and closed state. Elife 4.
  5. Koyama, Y., Hoshino, T., Tomizuka, N. and Furukawa, K. (1986). Genetic transformation of the extreme thermophile Thermus thermophilus and of other Thermus spp. J Bacteriol 166(1): 338-340.
  6. Lasa, I., Caston, J. R., Fernandez-Herrero, L. A., de Pedro, M. A. and Berenguer, J. (1992). Insertional mutagenesis in the extreme thermophilic eubacteria Thermus thermophilus HB8. Mol Microbiol 6(11): 1555-1564.
  7. Nakamura, A., Takakura, Y., Kobayashi, H. and Hoshino, T. (2005). In vivo directed evolution for thermostabilization of Escherichia coli hygromycin B phosphotransferase and the use of the gene as a selection marker in the host-vector system of Thermus thermophilus. J Biosci Bioeng 100(2): 158-163.
  8. Schwarzenlander, C. and Averhoff, B. (2006). Characterization of DNA transport in the thermophilic bacterium Thermus thermophilus HB27. FEBS J 273(18): 4210-4218.

简介

许多 物种港口基因组受到水平转移的签名的影响。 嗜热栖热菌 ( Tth )已被表征为天生胜任。本文所述的转化方案是基于在中指数期(OD <600> 0.3-0.4)处登记的最大DNA摄取速率。在这里我们描述了转化质粒和线性化基因组DNA的逐步方案,其中后者可以用作电穿孔以引入突变或在Thermus 分离物中产生基因缺失的替代方法,实例。

[背景] 嗜热栖热菌( Tth )是一种极端的嗜热菌种,由于其古老的系统发生起源,其蛋白质和大分子复合物的结晶的相对容易,以及其几种分离物的实验室生长条件下的快速生长和良好产量。在最常用的分离株中,菌株T。嗜热链球菌HB27和HB8构成最常用的模型,因为它们可以通过天然能力转化的高比率。任何来源和拓扑的DNA可以通过以约40kb/s /细胞的速率培养这些分离物的细胞容易地被吸收(Schwarzenlander和Averhoff,2006),显示约10个转化体/活细胞。为此,涉及在两种菌株的染色体(Averhoff,2009)中的五个基因座中编码的至少16种蛋白质的极性定位的天然竞争性装置(Gold等人,2015)基本上表达在组成型虽然转化效率在指数期较高。已经通过定向进化作为基因标记开发了编码热稳定变体的抗生素抗性基因的变体,以这种方式,可以在具有卡那霉素(Lasa等人,1992),潮霉素B Nakamura等人,2005)或博来霉素(Broun等人,2005)。链霉素抗性也可用于检查与同基因DNA的天然能力(Koyama等人,1986)。该协议描述了在指数生长期的培养物的高效转化,提供最大化转化效率的可重复数据。

材料和试剂

  1. 12 ml无菌塑料管
  2. 15 ml无菌塑料管
  3. 无菌塑料培养皿(标准尺寸; 100×15mm)
  4. 用于微量移液器的无菌吸头,5-200μl(例如,Daslab,目录号:162001)
  5. 用于微量移液器的无菌吸头,100-1,000μl(例如,Daslab,目录号:162222)
  6. 用于微量移液器的无菌吸头,10μl(例如,Metler Toledo,目录号:17004280)
  7. 例如HB27野生型(HB27/ATCC BAA-163/DSM 7039)的受体菌株 注意:其他嗜热栖热菌菌株如NAR1和HB8已经被定义为天然感受态,因此拥有转化所需的能力蛋白(Césaret al。,2011)。所有Thermus spp。此处提及的品系可在 DSMZ  收集。
  8. 硫酸卡那霉素(Sigma-Aldrich,目录号:K1377-5G)
  9. 胰蛋白胨(Conda,目录号:1612)
  10. 酵母提取物(Conda,目录号:1702)
  11. 氯化钠(NaCl)(EMD Millipore,目录号:1.06404.1000)
  12. 琼脂(A级)(Conda,目录号:1800)
  13. 水(富含碳酸气的矿泉水)
  14. DNA模板:纯化的质粒或基因组DNA **(含有热稳定的kat 盒)

  15. TB液体介质(参见配方)
  16. TB琼脂平板(见配方)

设备

  1. 50ml锥形瓶
  2. 旋转摇床培养箱达到70℃(150rpm)(Thermo Scientific,Thermo Scientific ,目录号:SHKE 420HP)
  3. 玻璃珠或吊具
  4. 高压釜设备(CertoClav,型号:EL 18L)
  5. 分光光度计(日立,型号:U-2000)
  6. 移液管P2,0.2-2μl(Gilson,Pipetman classic TM ,目录号:F144801)
  7. 移液管P20,2-20μl(Gilson,Pipetman classic TM ,目录号:F123600)
  8. 移液管P100,20-100μl(Gilson,Pipetman classic TM ,目录号:F123615)
  9. PipetteP200,50-200μl(Gilson,Pipetman classic TM ,目录号:F123601)
  10. 70℃加热器(Gemni,型号:Memmert UE400)
  11. 湿气室
    注意:优先使用Tupperware设备。加湿塑料袋也有效。加入纸巾,浸泡在Thermus水上并放置在室的底部将增强湿度维持。应避免通风,以保存湿度。下面的照片中提供了一个示例(图1)。


    图1.湿度室。用于孵育Thermus板的潮湿室的描述性照片。放置一块用Thermus水浸泡的纸和在腔室底部装有相同水的瓶盖以保持高湿度。

程序

  1. 培养物制备:待转化的主题菌株的生长(参见图2)。
    1. 在50ml锥形瓶中的10ml TB培养基中接种50μl饱和液体生长培养物(600nm处的光密度[OD 600] aprox。2)或从甘油储液或从冷冻颗粒。在后一种情况下,需要再增加一天才能达到最佳的培养条件
    2. 在65℃下在振荡培养箱中以150rpm培养过夜培养物
    3. 在50ml新鲜TB培养基中将过度生长的培养物稀释至OD 600 = 0.05,在50ml锥形瓶中。
      注意:
      1. 如果在过夜生长后培养物未达到饱和,强烈建议后续实验。
      2. 如果培养物在一夜之间没有最佳地生长并且应该进行实验,则可以达到0.07-0.08的起始OD <600>。
    4. 在65℃的振荡培养箱中孵育,直到达到0.3-0.4的OD <600。
      注意:建议接近0.3的OD 600


      图2. Thermus 转化方法的示意图。板显示100ng线性化质粒的转化的稀释,/em>盒,其侧翼为用于在HB27菌株中双重重组的两个基因组臂
  2. DNA添加
    1. 在无菌的12ml塑料管中加入0.01-1μgDNA(基因组〜0.01μg;质粒〜0.1-0.25μg; PCR产物〜1μg)。管可以在65℃预热。
      注意:
      1. 基因组DNA(携带集成在基因组中的选择性标记)不应当以更大的量添加,以避免重组比目标更远。
      2. 根据标准方案纯化DNA样品,并用水或TE洗脱。
      3. 预热管使培养的培养物的温度保持稳定。
    2. 向DNA中加入500μl指数生长培养物(见图2)
    3. 作为对照,准备15毫升没有DNA的塑料管,并加入500微升的指数生长培养物到这个管
  3. DNA转化
    1. 孵育每个管在振荡培养箱中在65°C下4小时
    2. 在选择性琼脂培养基上平板50μl和400μl细胞,通过使用玻璃珠或扩散器扩散 注意:
      1. 体积电镀取决于所采用的Thermus菌株的能力效率。以前的测定法电镀连续稀释点可以揭示适当的印版体积。
      2. 除了在非选择性琼脂培养基上电镀以外,还可以像其它培养基那样对其进行平板培养,因为它将作为活细胞的参考。
    3. 孵育板在湿室在65°C下48小时或直到菌落变得可见 注意:
      1. 通常,根据菌株的最佳生长来设定孵育温度,但是其应当适应于转化中使用的选择标记的限制(例如,赋予潮霉素抗性的hph5不能暴露于高于60℃的温度而卡那霉素在高于70℃的温度下可选择)。
      2. 转化产量高度依赖于所用的DNA模板和转化的菌株。例如,用100ng质粒DNA转化的HB27显示每个活细胞10-3个转化体的比率

数据分析

  1. 对于转化测定,应进行一组统计测试以比较和分析获得的转移频率。
  2. 转移频率计算为CFU转化体的数量除以活细胞的CFU的数量。为了检查转移频率之间的差异,可以使用Student's t检验和Mann-Whitney U检验。当比较测试几种DNA模板的菌株的可转化性时,可以通过单因素方差分析来解决各种DNA模板内和之间的差异的转移频率。如果测试不同的菌株,可以使用Wilcoxon检验来评估它们的转移频率之间的差异。可以实施简单的线性回归测试来建模DNA模板和转移频率之间的关系。 Post-hoc 在方便时应使用Tukey和Bonferroni测试。
  3. 至少应进行3次独立实验,以获得可靠且可重复的数据

笔记

  1. 该转化方案针对嗜热栖热菌(Thermus thermophilus)HB27进行优化,其显示在属内的最高天然能力效率和最高的DNA摄取率。具有较低能力的其他菌株应用更大量的DNA和/或更长的孵育期处理
  2. 在进行转化以引入突变的情况下,转化体应该在选择性平板上重划线,并在每种特定条件下进一步在液体中生长以确保稳定的突变。
  3. 所需的DNA量从使用基因组DNA时的10ng变化到使用PCR产物时的1,000ng。平均来说,使用100-250ng的复制质粒
  4. 不管使用的选择性标记,后孵育时间在65℃和150rpm下至少3至4小时。如果在指数期开始时添加DNA(OD 600 = 0.2),则时间延迟达5小时孵育。

食谱

  1. TB液体介质
    0.8%胰蛋白胨(w/v)
    0.4%酵母提取物(w/v)
    50mM NaCl,pH 7.5
    水(富含碳酸气的矿泉水)*
    pH可以通过加入NaOH调节
  2. TB琼脂平板
    0.8%胰蛋白胨(w/v)
    0.4%酵母提取物(w/v)
    50mM NaCl,pH 7.5
    1.8%琼脂(A级)(w/v) 水(富含碳酸气的矿泉水)*

注意:所用的弹簧矿泉水的分析组成(mg/ml)列在下面(表1)。可以使用易于购买的其他矿泉水,例如Evian水或类似此配方的实验室溶液。

表1.来自的泉水矿物成分 Jaraba Spring 在西班牙萨拉戈萨

致谢

这项工作由Grants BIO2013-44963-R和FP7-PEOPLE-2012-IAPP第324439号资助。A. Blesa与西班牙经济和竞争力部签订了一份FPI合同。

参考文献

  1. Averhoff,B。(2009)。  洗牌基因 FEMS Microbiol Rev 33(3):611-626。
  2. Broder,SJ,Wu,​​H。,Akerboom,J.,Turnbull,AP,de Vos,WM和van der Oost,J.(2005)。  设计用于超嗜热菌的可选择标记。

    J Biol Chem 280(12):11422-11431 。
  3. César,CE,Alvarez,L.,Bricio,C.,van Heerden,E.,Littauer,D。和Berenguer,J.(2011)。  极端嗜热细菌中的非常规侧向基因转移。 Int Microbiol 14(4):187-
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  5. Koyama,Y.,Hoshino,T.,Tomizuka,N.and Furukawa,K。(1986)。  极端嗜热菌Thermus thermophilus和其他Thermus 的遗传转化 em> 166(1):338-340。
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引用:Blesa, A. and Berenguer, J. (2016). Transformation of Thermus Species by Natural Competence. Bio-protocol 6(22): e2007. DOI: 10.21769/BioProtoc.2007.
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