搜索

Stationary-phase Mutagenesis Soft-agar Overlay Assays in Bacillus subtilis
枯草芽孢杆菌的软琼脂糖覆盖固相诱变   

引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Journal of Bacteriology
Feb 2017

Abstract

Elucidating how a population of non-growing bacteria generates mutations improves our understanding of phenomena like antibiotic resistance, bacterial pathogenesis, genetic diversity and evolution. To evaluate mutations that occur in nutritionally stressed non-growing bacteria, we have employed the strain B. subtilis YB955, which measures the reversions rates to the chromosomal auxotrophies hisC952, metB5 and leuC427 (Sung and Yasbin, 2002). This gain-of-function system has successfully allowed establishing the role played by repair systems and transcriptional factors in stress-associated mutagenesis (SPM) (Barajas-Ornelas et al., 2014; Gómez-Marroquín et al., 2016). In a recent study (Castro-Cerritos et al., 2017), it was found that Ribonucleotide Reductase (RNR) was necessary for SPM; this enzyme is essential in this bacterium. We engineered a conditional mutant of strain B. subtilis YB955 in which expression of the nrdEF operon was modulated by isopropyl-β-D-thiogalactopyranoside (IPTG) (Castro-Cerritos et al., 2017). The conditions to determine mutation frequencies conferring amino acid prototrophy in three genes (hisC952, metB5, leuC427) under nutritional stress in this conditional mutant are detailed here. This technique could be used to evaluate the participation of essential genes in the mutagenic processes occurring in stressed B. subtilis cells.

Keywords: Bacillus subtilis (枯草芽孢杆菌), Ribonucleotide reductase (核糖核苷酸还原酶), Amino acid starvation (氨基酸营养缺失), Stress-associated mutagenesis (压力相关诱变)

Background

Around 270 genes, including dnaA, dnaB, dnaC, involved in DNA replication as well as the nrdEF operon, which encodes RNR, are considered essential for B. subtilis growth (Kobayashi et al., 2003). Here we describe a protocol that has been applied to understand the role of this enzyme in modulating events of mutagenesis in nutritionally stressed non-growing cells of strain B. subtilis YB955 (hisC952, lecuC427, metB5). To this end, we implemented a genetic system that represses the expression of the essential nrdEF operon (non-permissive condition) while limiting two of the essential amino acids to avoid transient and weak phenotypes produced by variants of less efficient enzymes (Pybus et al., 2010). These restriction conditions allowed us first inquiring whether RNR influences mutagenesis in a population of non-growing (but viable) B. subtilis cells. In a second stage, a soft agar overlay, which provides permissive conditions for growth, allows detection of prototrophic colonies only if a mutation that restores at least one amino acid prototrophy has occurred. Whereas this protocol has been employed here with RNR, it can be potentially extended to study additional genes that are essential for metabolism as well as DNA replication and transcription. In addition, conditions may be adjusted to other bacterial species or selective markers.

Materials and Reagents

  1. 15- and 50-ml sterile Falcon tubes. Conical Centrifuge Tubes (Corning, Axygen®, catalog numbers: SCT-15ML-500 ; SCT-50ML-500 )
  2. Pasteur pipettes
  3. Disposable (90 mm diameter) Petri dishes (BD, catalog number: 252777 )
  4. Cellulose filter paper 0.22 µm (Merck, catalog number: GSWP04700 )
  5. Bacillus subtilis strains YB955 (hisC952 metB5 leuC427 xin-1 Spβs) and PERM1017 (hisC952 metB5 leuC427 ΔnrdE::lacZ Pspac-nrdEF::ery Eryr)
  6. Penassay broth (PAB; antibiotic medium 3; Difco Laboratories, Sparks, MD) (Fisher Scientific, catalog number: DF0243-17-8)
    Manufacturer: BD, catalog number: B224320 .
  7. Isopropyl-β-D-thiogalactoside (IPTG) (Promega, catalog number: V3951 )
  8. L-Leucine (Sigma-Aldrich, catalog number: L8000 )
  9. L-Glutamic acid (Sigma-Aldrich, catalog number: 49449 )
  10. L-Isoleucine (Sigma-Aldrich, catalog number: 58879 )
  11. L-Methionine (Sigma-Aldrich, catalog number: M9625 )
  12. L-Histidine monohydrochloride monohydrate (Sigma-Aldrich, catalog number: 53370 )
  13. Ammonium sulfate ((NH4)2SO4) (Karal, catalog number: 3016 )
  14. Potassium phosphate dibasic (K2HPO4) (Karal, catalog number: 5080 )
  15. Potassium phosphate monobasic (KH2PO4) (Karal, catalog number: 5079 )
  16. Sodium citrate (C6H5Na3O7·2H2O) (Avantor Performance Materials, J.T. Baker®, catalog number: 3646-01 )
  17. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Karal, catalog number: 6056 )
  18. Magnesium chloride hexahydrate (MgCl2·6H2O) (Karal, catalog number: 6054 )
  19. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C2536 )
    Note: This product has been discontinued.
  20. Ferrous chloride tetrahydrate (FeCl2·4H2O) (Avantor Performance Materials, J.T. Baker®, catalog number: 2064-01 )
  21. Zinc chloride (ZnCl2) (Sigma-Aldrich, catalog number: Z0152 )
  22. Copper(II) chloride dihydrate (CuCl2·2H2O) (Karal, catalog number: 8021 )
  23. Cobalt(II) chloride hexahydrate (CoCl2·6H2O) (Karal, catalog number: 8026 )
  24. Sodium molybdate dehydrate (Na2MoO4·2H2O) (Karal, catalog number: 4072 )
  25. Agar (BD, catalog number: 215000 )
  26. Dextrose (BD, catalog number: 216800 )
  27. PAB medium (PAB) (see Recipes)
  28. Amino acid solutions (see Recipes)
  29. 1 M IPTG (see Recipes)
  30. 10x Spizizen salts (10x SS)
  31. 100x trace elements (see Recipes)
  32. Spizizen minimal salts (SS) (see Recipes)
  33. Spizizen minimal medium (SMM) (see Recipes)
  34. Soft agar (see Recipes)

Equipment

  1. Orbital shaker Lab-Line MaxQ 4000 (Thermo Fisher Scientific, Thermo ScientificTM, model: MaxQTM 4000 )
  2. Conventional incubator set at 37 °C
  3. Spectrophotometer (Biochrom, model: WPA CO7500 )
  4. Centrifuge Thermo Scientific IEC CL30R (Thermo Fisher Scientific, Thermo ScientificTM, model: IEC CL30R )
  5. Conventional autoclave
  6. 125-ml sterile Erlenmeyer flasks
  7. Reusable syringe filter holders (Merck, catalog number: SX0002500 )
  8. Magnetic stirring bar

Procedure

  1. Cell propagation
    1. Propagate a 1:50 dilution of an overnight culture of B. subtilis strains in 25 ml of PAB medium (supplemented with 0.1 mM IPTG, if necessary, see Recipes). Incubate at 37 °C with vigorous shaking (250 rpm), monitor growth by taking periodical readings on a spectrophotometer set at 600 nm.
    2. Plot OD600 nm versus time in a semi-log sheet and identify time zero (T0), defined as the time point in the culture when the slopes of the logarithmic and stationary phases of growth intercept (see Supplemental Figure S1).
    3. 90 min after T0, transfer 10 ml of the culture to a sterile 15-ml Falcon tube and centrifuge (4, 800 x g; 10 min, 23 °C); wash cell pellets 3 times each with 10 ml of 1x SS (see Recipes) and resuspend in an equal volume of the same solution.

  2. Viable counts
    Determine the initial number of bacteria for each strain by preparing serial ten-fold dilutions in 1x SS, plating dilutions 10-5 to 10-7 on SMM supplemented with histidine, methionine and leucine, incubating for 48 h at 37 °C and scoring the number of CFU per ml.

  3. Selective pressure
    1. Take aliquots of 1 ml of the cell suspension and, independently, transfer to three 50-ml Falcon tubes containing 49 ml of soft agar SA (one tube with SA1 and two for SA2) pre-warmed at 42 °C.
    2. Decant 4 ml of the final suspension onto each of 12 plates of SMM-His-Leu- and 24 plates of SMM-Met-Leu-, allow to solidify and incubate the plates at 37 °C for 10 days. Of note, supplement plates with 1 mM IPTG to activate nrdEF expression in strain B. subtilis PERM1017.
      Note: The combination (His-, Met-) in addition to the absence of three amino acids (His-, Leu-, Met-) is not used to avoid favor suppressor mutations that have been suggested revert His and Met auxotrophies simultaneously (Sung and Yasbin, 2002).
    Note: Soft agar for inoculation (SA) helps to maintain cells immobilized; thus, avoiding the spread of colonies during Procedure D (colony growth).

  4. Colony growth
    1. During days 2, 4, 6, 8, and 10, pick two plates of His- Leu- medium and four plates of Met- Leu-, proceed to prepare soft agar to obtain His- (SA3), Met- (SA4) or Leu- (SA5) media (see Recipes). Over these SMM plates pour a first overlay with the cells and a second overlay with the selective medium (Figure 1 and Supplemental Figure S2). This results in single amino acid dropout media that selects for His+, Met+ or Leu+ revertants. Incubate the plates at 37 °C and record the number of revertants His+, Met+, and Leu+ colonies that arise after 48 h of incubation.
    2. Determine the average number of revertants from the two plates used for each selective media at the respective day of incubation; calculate the mutation frequency using the accumulated number of revertants and the number of CFU plated.


      Figure 1. Procedure for Stationary-phase mutagenesis soft-agar overlay assays. A. Cell propagation. Cultivate Bacillus subtilis strains PERM1017, YB955 and PERM1202 in PAB medium until 90 min after the end of exponential growth; wash with 1x SS. B. Viable counts, colony forming units are determined by serial ten-fold dilutions. C. Selective pressure. Cells are plated on selective medium using the corresponding soft agar overlay (SA1 or SA2) and incubated for 10 days at 37 °C. D. Colony growth. A second soft agar overlay (SA3, SA4 or SA5) is added to a set of plates of each selective medium to restore one of the missing amino acids, revertant His+, Met+ and Leu+ colonies are scored after 48 h of incubation. E. Cell survival is monitored during days 2, 4, 6, 8, and 10 from selection SMM-His-Leu- and SMM-Met-Leu- plates as described in Procedure E. In case of strain PERM1017, media must be supplemented with 1 mM IPTG, as indicated in the procedure (perform this procedure on days 2, 4, 6, 8 and 10) (Pybus et al., 2010).

  5. Cell survival
    1. Determine the abilities of strains with different genotypes to survive in SMM-His-Leu- or SMM-Met-Leu- plates during the ten days of the experiment.
    2. Employing sterile Pasteur pipettes, during days, 2, 4, 6, and 8, remove three-agar plugs from areas free of colonies of media plates employed for ‘selective pressure’ (Procedure C). This procedure allows quantifying the initially plated fraction of non-revertant cells that have survived the incubation period.
    3. Suspend the plugs in 400 µl of 1x SS, vortex thoroughly, plate serial ten-dilutions on SMM containing all the essential amino acids (50 μg/ml). Score the number of colonies after 48 h of incubation at 37 °C.

Note: A representative example is presented in Table S1. The data are from an experiment of SPM using soft agar overlay for the hisC allele in strain B. subtilis YB955.

Data analysis

Following this procedure, Figure 2 shows the accumulation of His+, Met+ and Leu+ revertants in the parental strain YB955 as well as in the conditional RNR strain (PERM1017), under conditions that induce (+ IPTG) or not (- IPTG) the expression of the nrdEF operon.


Figure 2. Frequencies of stationary-phase reversions to His+ (A), Met+ (B) and Leu+ (C) of strains B. subtilis YB955 (gray squares), PERM1017 [(conditional RNR strain) (+ IPTG; black squares)] PERM1017 (- IPTG; white squares) were determined as described in procedures. Data represent counts averaged from two separate tests ± standard deviation, Data presented for strains YB955 and PERM1017 (- IPTG) were reported in: Castro-Cerritos, K. V., Yasbin, R. E., Robleto, E. A. and Pedraza-Reyes, M. (2017). Role of ribonucleotide reductase in Bacillus subtilis stress-associated mutagenesis. J Bacteriol 199(4).

Recipes

  1. PAB medium (PAB)
    Dissolve 1.75 g of PAB medium in 100 ml dH2O, autoclave at 121 °C for 15 min
  2. Amino acid solutions [5 mg/ml]
    Dissolve 250 mg of the required amino acid in 50 ml of ddH2O
    Sterilize by filtration
  3. 1 M IPTG
    Dissolve 1.19 g of IPTG in 5 ml of ddH2O and sterilize by filtration
  4. 10x Spizizen salts (10x SS)
    Dissolve:
    10 g (NH4)2SO4
    70 g K2HPO4
    30 g KH2PO4
    5 g sodium citrate·2H2O
    1 g MgSO4·7H2O
    Add ddH2O to 500 ml
    Autoclave at 121 °C for 15 min
  5. 100x trace elements
    Dissolve:
    1.28 g MgCl2·6H2O
    0.055 g CaCl2
    0.135 g FeCl2·6H2O
    0.01 g ZnCl2
    0.0043 g CuCl2·2H2O
    0.006 g CoCl2·6H2O
    0.006g Na2MoO4·2H2O
    Add ddH2O to 100 ml
    Autoclave at 121 °C for 15 min
  6. Spizizen minimal medium (SMM)
    In 1 L Erlenmeyer flask:
    Add 7.5 g of agar to 430 ml of distilled water, add a magnetic stirring bar
    Autoclave at 121 °C for 15 min
    Add 50 ml of sterile 10x SS
    Cool to 50-55 °C under constant stirring
    Add 5 ml of 0.5 g/ml dextrose and a mixture of the following amino acid solutions under constant stirring:
    1. For medium supplementing histidine, methionine, and leucine:
      5 ml of 5 mg/ml isoleucine
      5 ml of 5 mg/ml glutamic acid
      5 ml of 5 mg/ml methionine
      5 ml of 5 mg/ml histidine
      5 ml of 5 mg/ml leucine
    2. For medium lacking histidine and leucine (SMM-His-Leu-):
      5 ml of 5 mg/ml isoleucine
      5 ml of 5 mg/ml glutamic acid
      5 ml of 5 mg/ml methionine
      20 µl of 5 mg/ml histidine
      20 µl of 5 mg/ml leucine
    3. For medium lacking methionine and leucine (SMM-Met-Leu-):
      5 ml of 5 mg/ml isoleucine
      5 ml of 5 mg/ml glutamic acid
      5 ml of 5 mg/ml histidine
      20 µl of 5 mg/ml methionine
      20 µl of 5 mg/ml leucine
    Note: For strain B. subtilis PERM1017, supplement the three media with 1 mM IPTG.
    Pour:
    1. 4.0 ml of SMM-His-Leu- into 12 (90 x 15 mm) Petri dishes
    2. 4.0 ml of SMM-Met-Leu- into 24 (90 x 15 mm) Petri dishes
  7. Soft agar medium (SMA) for cell inoculation
    1. 0.7% agar
      Add 1.4 g agar to 200 ml of water in a 1 L Erlenmeyer flask with a magnetic stirring bar, autoclave at 121 °C for 15 min.
    Note: In 50 ml Falcon tubes prepare the following mixtures:
    1. For medium lacking histidine and leucine (His- Leu- medium; SA1):
      5 ml 10x SS
      500 µl of 5 mg/ml isoleucine
      500 µl of 5 mg/ml glutamic acid
      500 µl of 5 mg/ml methionine
      500 µl of 0.5 g/ml dextrose
    2. For medium lacking methionine and leucine (Met- Leu- medium; SA2):
      5 ml 10x SS
      500 µl 5 mg/ml isoleucine
      500 µl 5 mg/ml glutamic acid
      500 µl of 5 mg/ml histidine
      500 µl 0.5 g/ml dextrose
    Notes:
    1. Supplement the media with 1 mM IPTG in the case of B. subtilis PERM1017.
    2. Add pre-warmed agar (50-55 °C) to a final volume of 50 ml in each tube; mix gently.
  8. Soft agar to obtain His-, Met- or Leu- media
    1. Add 350 mg agar to 50 ml of water in a flask with a magnetic stirrer bar, autoclave at 121 °C for 15 min.
    Note: Prepare the following mixtures in 15 ml Falcon tubes:
    1. For His- medium (SA3):
      1 ml of 10x SS
      1 ml of 5 mg/ml leucine
      100 µl of 5 mg/ml isoleucine
      100 µl of 5 mg/ml glutamic acid
      100 µl of 5 mg/ml methionine
      100 µl of 0.5 g/ml dextrose 
    2. For Met- medium (SA4):
      1 ml of 10x SS
      1 ml of 5 mg/ml histidine
      100 µl of 5 mg/ml isoleucine
      100 µl of 5 mg/ml glutamic acid
      100 µl of 5 mg/ml leucine
      100 µl of 0.5 g/ml dextrose
    3. For Leu- medium (SA5):
      1 ml 10x SS
      1 ml of 5 mg/ml methionine
      100 µl of 5 mg/ml isoleucine
      100 µl of 5 mg/ml glutamic acid
      100 µl of 5 mg/ml histidine
      100 µl of 0.5 g/ml dextrose
    Notes:
    1. In the case of B. subtilis PERM1017, 100 µl of 1 M IPTG must be added to the mixtures to reach a final concentration of 1 mM (considering a final volume of 50 ml in the Petri dish).
    2. Add pre-warmed agar (50-55 °C) to a final volume of 10 ml in each tube; mix gently.

Acknowledgments

This work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT; Grants 205744 and 221231) of México and by the University of Guanajuato (Grants 936-2016 and 1090-2016) to M.P-R. Work at ER was supported by the NIH grant (R15GM110624). KV. C-C and N. V-N were supported by scholarships from CONACYT. The authors declare no conflict of interest.

References

  1. Barajas-Ornelas R del, C., Ramirez-Guadiana, F. H., Juarez-Godinez, R., Ayala-Garcia, V. M., Robleto, E. A., Yasbin, R. E. and Pedraza-Reyes, M. (2014). Error-prone processing of apurinic/apyrimidinic (AP) sites by PolX underlies a novel mechanism that promotes adaptive mutagenesis in Bacillus subtilis. J Bacteriol 196(16): 3012-3022.
  2. Castro-Cerritos, K. V., Yasbin, R. E., Robleto, E. A. and Pedraza-Reyes, M. (2017). Role of ribonucleotide reductase in Bacillus subtilis stress-associated mutagenesis. J Bacteriol 199(4).
  3. Gómez-Marroquín, M., Martin, H. A., Pepper, A., Girard, M. E., Kidman, A. A., Vallin, C., Yasbin, R. E., Pedraza-Reyes, M. and Robleto, E. A. (2016). Stationary-phase mutagenesis in stressed Bacillus subtilis cells operates by Mfd-dependent mutagenic pathways. Genes (Basel) 7(7).
  4. 5. Kobayashi, K., Ehrlich, S. D., Albertini, A., Amati, G., Andersen, K. K., Arnaud, M., Asai, K., Ashikaga, S., Aymerich, S., Bessieres, P., Boland, F., Brignell, S. C., Bron, S., Bunai, K., Chapuis, J., Christiansen, L. C., Danchin, A., Debarbouille, M., Dervyn, E., Deuerling, E., Devine, K., Devine, S. K., Dreesen, O., Errington, J., Fillinger, S., Foster, S. J., Fujita, Y., Galizzi, A., Gardan, R., Eschevins, C., Fukushima, T., Haga, K., Harwood, C. R., Hecker, M., Hosoya, D., Hullo, M. F., Kakeshita, H., Karamata, D., Kasahara, Y., Kawamura, F., Koga, K., Koski, P., Kuwana, R., Imamura, D., Ishimaru, M., Ishikawa, S., Ishio, I., Le Coq, D., Masson, A., Mauel, C., Meima, R., Mellado, R. P., Moir, A., Moriya, S., Nagakawa, E., Nanamiya, H., Nakai, S., Nygaard, P., Ogura, M., Ohanan, T., O'Reilly, M., O'Rourke, M., Pragai, Z., Pooley, H. M., Rapoport, G., Rawlins, J. P., Rivas, L. A., Rivolta, C., Sadaie, A., Sadaie, Y., Sarvas, M., Sato, T., Saxild, H. H., Scanlan, E., Schumann, W., Seegers, J. F., Sekiguchi, J., Sekowska, A., Seror, S. J., Simon, M., Stragier, P., Studer, R., Takamatsu, H., Tanaka, T., Takeuchi, M., Thomaides, H. B., Vagner, V., van Dijl, J. M., Watabe, K., Wipat, A., Yamamoto, H., Yamamoto, M., Yamamoto, Y., Yamane, K., Yata, K., Yoshida, K., Yoshikawa, H., Zuber, U. and Ogasawara, N. (2003). Essential Bacillus subtilis genes. Proc Natl Acad Sci U S A 100(8): 4678-4683.
  5. Pybus, C., Pedraza-Reyes, M., Ross, C. A., Martin, H., Ona, K., Yasbin, R. E. and Robleto, E. (2010). Transcription-associated mutation in Bacillus subtilis cells under stress. J Bacteriol 192(13): 3321-3328.
  6. Sung, H. M. and Yasbin, R. E. (2002). Adaptive, or stationary-phase, mutagenesis, a component of bacterial differentiation in Bacillus subtilis. J Bacteriol 184(20): 5641-5653.

简介

阐明非增长细菌群体如何产生突变提高了我们对诸如抗生素抗性,细菌发病机理,遗传多样性和进化等现象的理解。为了评估在营养强调的非生长细菌中发生的突变,我们使用了菌株B。它们测量对染色体营养缺陷型hisC952,metB5和leuC427(Sung和Yasbin,2002)的逆转速率。这种功能获得性系统已成功地允许建立修复系统和转录因子在压力相关的诱变(SPM)中所起的作用(Barajas-Ornelas等人,2014;Gómez-Marroquín等。,2016)。在最近的研究(Castro-Cerritos等人,2017)中,发现核糖核苷酸还原酶(RNR)是SPM所必需的;这种酶在这种细菌中是必不可少的。我们设计了菌株B的条件性突变体。其中通过异丙基-β-D-硫代半乳糖吡喃糖苷(IPTG)(Castro-Cerritos等人,2017)调控了nrdEF操纵子的表达, 。确定在该条件突变体中在营养胁迫下确定三个基因(hisC952,metB5,leuC427)中的氨基酸原养型的突变频率的条件详细这里。这种技术可以用来评估重要基因在应激B发生的致突变过程中的参与。枯草杆菌细胞。

【背景】涉及DNA复制的大约270个基因,包括dnaA,dnaB,dnaC和nrdEF操纵子,它编码RNR,被认为是必不可少的。枯草芽孢杆菌生长(Kobayashi等人,2003)。在这里我们描述了一个协议,已被用来了解这种酶的作用调节事件诱变事件营养应激的非生长细菌菌株枯草芽孢杆菌YB955(emC952,emCecuC427, em>, metB5 )。为此,我们实施了抑制基本操纵子(非允许条件)的表达的遗传系统,同时限制两种必需氨基酸以避免由变体产生的短暂和弱表型高效的酶(Pybus等人,2010)。这些限制条件使我们首先询问RNR是否影响非生长(但可行)B群体中的诱变。枯草杆菌细胞。在第二阶段,提供允许生长条件的软琼脂覆盖物允许只有在发生恢复至少一个氨基酸原养型的突变的情况下才能检测原养型菌落。尽管这个方案已经在RNR中使用,但它可以扩展到研究代谢以及DNA复制和转录所必需的其他基因。另外,条件可以调整为其他细菌种类或选择性标记。

关键字:枯草芽孢杆菌, 核糖核苷酸还原酶, 氨基酸营养缺失, 压力相关诱变

材料和试剂

  1. 15和50毫升无菌猎鹰管。锥形离心管(Corning,Axygen ,目录号:SCT-15ML-500; SCT-50ML-500)
  2. 巴斯德移液器
  3. 一次性(90毫米直径)培养皿(BD,目录号:252777)
  4. 纤维素滤纸0.22μm(Merck,目录号:GSWP04700)
  5. 将枯草芽孢杆菌菌株YB955(hisC952 metB5 leuC427xin-1spββsiem)和PERM1017( hisC952 metB5 leuC427 n nrdE :: lacZ spac-nrdEF :: ery Ery r )
  6. Penassay肉汤(PAB;抗生素培养基3; Difco Laboratories,Sparks,MD)(Fisher Scientific目录号:DF0243-17-8)
    制造商:BD,目录号:B224320。
  7. 异丙基-β-D-硫代半乳糖苷(IPTG)(Promega,目录号:V3951)
  8. L-亮氨酸(Sigma-Aldrich,目录号:L8000)
  9. L-谷氨酸(Sigma-Aldrich,目录号:49449)
  10. L-异亮氨酸(Sigma-Aldrich,目录号:58879)
  11. L-甲硫氨酸(Sigma-Aldrich,目录号:M9625)
  12. L-组氨酸单盐酸盐一水合物(Sigma-Aldrich,目录号:53370)
  13. 硫酸铵((NH 4)2 SO 4)(Karal,目录号:3016)
  14. 磷酸二氢钾(K 2 HPO 4)(Karal,目录号:5080)
  15. 磷酸二氢钾(KH 2 PO 4)(Karal,目录号:5079)
  16. 柠檬酸钠(C 6 H 5)Na 3 O 7•2H 2 O )(Avantor Performance Materials,JT Baker ®,目录号:3646-01)
  17. 硫酸镁七水合物(MgSO4•7H2O)(Karal,目录号:6056)
  18. 氯化镁六水合物(MgCl 2•6H 2 O)(Karal,目录号:6054)
  19. 氯化钙二水合物(CaCl 2•2H 2 O)(Sigma-Aldrich,目录号:C2536)
    注:此产品已停产。
  20. 氯化亚铁四水合物(FeCl 2•4H 2 O)(Avantor Performance Materials,JT Baker,目录号:2064-01) />
  21. 氯化锌(ZnCl 2)(Sigma-Aldrich,目录号:Z0152)
  22. 氯化铜(II)二水合物(CuCl 2•2H 2 O)(Karal,目录号:8021)
  23. 氯化钴(II)六水合物(CoCl 2•6H 2 O)(Karal,目录号:8026)
  24. 脱水钼酸钠(Na 2 MoO 4•2H 2 O)(Karal,目录号:4072)
  25. 琼脂(BD,目录号:215000)
  26. 葡萄糖(BD,目录号:216800)
  27. PAB培养基(PAB)(见食谱)
  28. 氨基酸溶液(见食谱)
  29. 1 M IPTG(见食谱)
  30. 10倍Spizizen盐(10倍不锈钢)
  31. 100倍微量元素(见食谱)
  32. Spizizen最低盐(SS)(见食谱)
  33. Spizizen基本培养基(SMM)(见食谱)
  34. 软琼脂(见食谱)

设备

  1. 轨道振荡器Lab-Line MaxQ 4000(Thermo Fisher Scientific,Thermo Scientific TM,型号:MaxQ TM 4000)
  2. 传统的培养箱设置在37°C
  3. 分光光度计(Biochrom,型号:WPA CO7500)
  4. 离心机Thermo Scientific IEC CL30R(Thermo Fisher Scientific,Thermo Scientific TM,型号:IEC CL30R)
  5. 常规高压灭菌器

  6. 125毫升无菌锥形瓶
  7. 可重复使用的注射器过滤器支架(Merck,产品目录号:SX0002500)
  8. 磁力搅拌棒

程序

  1. 细胞繁殖
    1. 繁殖1:50稀释的过夜培养物。在25ml的PAB培养基(补充有0.1mM IPTG,如果需要的话,参见食谱)中培养枯草芽孢杆菌菌株。剧烈摇动(250转/分钟)37°C孵育,通过设置在600纳米的分光光度计定期读数监测生长。
    2. 在半对数表中绘制OD 600nm与时间的关系曲线,并确定时间零点(T 0),定义为培养时间点,当对数和增长拦截的固定阶段(请参阅补充图S1 ) 。
    3. 在T 0分钟后90分钟,将10ml培养物转移到无菌的15ml Falcon管中并离心(4,800×g; 10分钟,23℃)。每次用10ml 1x SS洗涤细胞沉淀3次(见食谱),并重新悬浮在相同体积的同一溶液中。

  2. 可行的计数
    通过在1×SS中制备系列十倍稀释液,在补充有组氨酸,甲硫氨酸的SMM上的10 -5至10 -7的平板接种稀释液中确定每种菌株的初始细菌数量和亮氨酸,在37℃孵育48小时,并计算每毫升CFU的数量。

  3. 选择压力
    1. 取1毫升细胞悬液的等分试样,并独立地转移到含有49毫升软琼脂SA(一个SA1和两个SA2)的50ml Falcon管中,预热到42℃。
    2. 将4ml的最终混悬液分别注射到12个SMM-His-Leu-2平板和24个SMM-Met-Leu-允许固化并在37℃孵育平板10天。值得注意的是,用1mM IPTG补充平板以在菌株B中激活 nrdEF 表达。枯草杆菌 PERM1017。
      除了缺少三个氨基酸(His - ,Leu > , - )不用于避免有利的抑制突变同时还原His和Met营养缺陷(Sung和Yasbin,2002)。
    注意:接种软琼脂(SA)有助于维持细胞固定;从而避免程序D(菌落生长)期间菌落扩散。

  4. 菌落生长
    1. 在第2,4,6,8和10天期间,挑选两个His-Leu-Medium培养板和四个Met-Leu-Leu培养板(SA3),Met(SA4)或Leu - ,进行软琼脂准备以获得His- >(SA5)媒体(请参阅食谱)。在这些SMM板上倒出第一个覆盖细胞,第二个覆盖的选择性培养基(图1和图2)。 docx“target =”_ blank“>补充图S2 )。这导致单氨基酸丢失介质选择His +,Met +或Leu +回复体。在37℃孵育平板,并记录48小时后出现的回复突变体His +,Met +,和Leu + +集落的数目孵化。
    2. 确定在各自选择性培养基中使用的两个平板在各自培养日的平均回复物数量;
      使用累计回复次数和CFU数量来计算突变频率

      图1.固定相诱变软琼脂覆盖测定的步骤A.细胞繁殖。在PAB培养基中培养枯草芽孢杆菌菌株PERM1017,YB955和PERM1202,直到指数生长结束后90分钟;用1x SS洗。 B.可行的计数,菌落形成单位通过连续十倍稀释确定。 C.选择压力。使用相应的软琼脂覆盖物(SA1或SA2)将细胞铺在选择性培养基上并在37℃孵育10天。 D.殖民地增长。将第二个软琼脂覆盖层(SA3,SA4或SA5)加入到每个选择性培养基的一组平板中以恢复缺失的氨基酸之一,回复His +,Met +在培养48小时后对Leu +菌落进行评分。 E.在第2,4,6,8和10天期间从选择SMM-His-Leu-2和SMM-Met-2进行细胞存活的监测。如程序E中所述。在菌株PERM1017的情况下,培养基必须补充有1mM IPTG,如程序中所示(在第2,4,6,8,10天, 8和10)(Pybus et al。,2010)。

  5. 细胞存活
    1. 确定具有不同基因型的菌株在SMM-His-Leu-sup或SMM-Met-Leu-在实验的十天期间,
    2. 使用无菌巴斯德吸液管,在第2,4,6和8天,从用于“选择性压力”(程序C)的培养基平板的无菌区域移除三个琼脂塞。这个程序允许定量在潜伏期存活的非回复细胞的初始铺板部分。
    3. 将塞子悬浮于400μl1x SS中,彻底涡旋,在含有所有必需氨基酸(50μg/ ml)的SMM上进行系列十倍稀释。
      在37°C孵育48小时后,对菌落数进行评分
注意:一个具有代表性的例子在 Table S1 。这些数据来自SPM的实验,使用软琼脂覆盖菌株枯草芽孢杆菌YB955中的hisC等位基因。

数据分析

在此过程之后,图2显示了在亲代菌株YB955中的His +,Met +和Leu + +回复物的积累,以及在条件性RNR菌株(PERM1017),在诱导(+ IPTG)或不(IPTG)条件下,nrdEF操纵子的表达。


(A),Met(强),强(强),强(强) (B)和Leu + (C) EM>的 B中。如方法中所述测定YB955(灰色方块),PERM1017 [(条件RNR品系)(+ IPTG;黑色方块)] PERM1017(-IPTG;白色方块)数据代表来自两个单独测试的平均数±标准差,对于菌株YB955和PERM1017(-IPTG)所呈现的数据报道于:Castro-Cerritos,KV,Yasbin,RE,Robleto,EA和Pedraza-Reyes,M. 2017年)。 核糖核苷酸还原酶的作用 枯草芽孢杆菌 “压力相关的诱变”。J Bacteriol 199(4)。

食谱

  1. PAB培养基(PAB)
    将1.75g PAB培养基溶于100ml dH 2 O,121℃高压灭菌器中15分钟。
  2. 氨基酸溶液[5毫克/毫升]
    将250mg所需氨基酸溶于50ml ddH 2 O中 通过过滤消毒
  3. 1 M IPTG
    将1.19g IPTG溶解于5ml ddH 2 O中并通过过滤灭菌。
  4. 10倍Spizizen盐(10倍不锈钢)
    解散:
    10克(NH 4)2 SO 4 4/4> 70克K 2 HPO 4 4克/克 30克KH 2 PO 4 4克/克 5克柠檬酸钠•2H 2 O
    1克MgSO 4•7H 2 O 0 加入ddH 2 O至500毫升
    在121°C高压灭菌15分钟
  5. 100x微量元素
    解散:
    1.28克MgCl2•6H2O 2 O. 0.055克CaCl 2 2/2 0.135克FeCl 2•6H 2 O 0 0.01克ZnCl 2 2/2 0.0043克CuCl 2•2H 2 O 0 0.006克CoCl 2•6H 2 O 0 0.006g Na 2 MoO 4•2H 2 O
    加入ddH 2 O至100毫升
    在121°C高压灭菌15分钟
  6. Spizizen基本介质(SMM)
    在1升锥形瓶中:
    将7.5克琼脂加入430毫升蒸馏水中,加一个磁力搅拌棒
    在121°C高压灭菌15分钟
    加入50毫升无菌10x SS
    在恒定搅拌下冷却至50-55°C 在恒定的搅拌下加入5ml 0.5g / ml右旋糖和下列氨基酸溶液的混合物:
    1. 对于培养基补充组氨酸,甲硫氨酸和亮氨酸:
      5毫升5毫克/毫升异亮氨酸
      5毫升5毫克/毫升谷氨酸
      5毫升5毫克/毫升甲硫氨酸
      5毫升5毫克/毫升组氨酸
      5毫升5毫克/毫升亮氨酸
    2. 对于缺乏组氨酸和亮氨酸(SMM-His→Leu→ - )的培养基来说:
      5毫升5毫克/毫升异亮氨酸
      5毫升5毫克/毫升谷氨酸
      5毫升5毫克/毫升甲硫氨酸
      20μl5mg / ml组氨酸
      20μl的5mg / ml亮氨酸
    3. 对于缺乏甲硫氨酸和亮氨酸(SMM-Met-Leu-)的培养基来说:
      5毫升5毫克/毫升异亮氨酸
      5毫升5毫克/毫升谷氨酸
      5毫升5毫克/毫升组氨酸
      20微升5毫克/毫升甲硫氨酸
      20μl的5mg / ml亮氨酸
    注:对于菌株枯草芽孢杆菌PERM1017,用1mM IPTG补充三种培养基。
    倒:
    1. 4.0毫升的SMM-His-Leu-2分成12个(90×15毫米)培养皿。
    2. 在24(90×15mm)培养皿中加入4.0ml SMM-Met-Leu-
  7. 用于细胞接种的软琼脂培养基(SMA)
    1. 0.7%琼脂
      在一个带磁力搅拌棒的1L锥形瓶中加入1.4g琼脂到200ml水中,121℃高压灭菌15分钟。
    注意:在50毫升猎鹰管准备下列混合物:
    1. 对于培养基中缺乏组氨酸和亮氨酸(His→Leu→中等; SA1):
      5毫升10x SS
      500μl的5mg / ml异亮氨酸
      500微升5毫克/毫升的谷氨酸
      500微升5毫克/毫升甲硫氨酸
      500微升的0.5克/毫升葡萄糖
    2. 对于缺乏甲硫氨酸和亮氨酸(Met-Leu-Medium,SA2)的培养基:
      5毫升10x SS
      500μl5mg / ml异亮氨酸
      500微升5毫克/毫升谷氨酸
      500微升5毫克/毫升组氨酸
      500μl0.5μg/ ml右旋糖
    注意:
    1. 在枯草芽孢杆菌PERM1017的情况下,用1mM IPTG补充培养基。
    2. 将预热的琼脂(50-55℃)加入每个管中的最终体积为50ml;轻轻搅拌。
  8. 软琼脂可以获得His-sup,Met-sup或Leu-sup-media

    1. 在磁力搅拌棒的烧瓶中加入350毫克琼脂到50毫升水中,121℃高压灭菌15分钟。
    注意:在15ml Falcon管中准备以下混合物:
    1. 对于他的 - 中等(SA3):
      1毫升的10倍SS
      1毫升5毫克/毫升亮氨酸
      100μl的5mg / ml异亮氨酸
      100微升5毫克/毫升谷氨酸
      100微升5毫克/毫升甲硫氨酸
      100μl的0.5g / ml右旋糖
    2. Met - 中(SA4):
      1毫升的10倍SS
      1毫升5毫克/毫升组氨酸
      100μl的5mg / ml异亮氨酸
      100微升5毫克/毫升谷氨酸
      100μl的5毫克/毫升亮氨酸
      100微升0.5克/毫升右旋糖
    3. 对于Leu - 中等(SA5):
      1毫升10x SS
      1毫升5毫克/毫升甲硫氨酸
      100μl的5mg / ml异亮氨酸
      100微升5毫克/毫升谷氨酸
      100μl的5mg / ml组氨酸
      100μl的0.5g / ml右旋糖
    注意:
    1. 在枯草芽孢杆菌PERM1017的情况下,必须向混合物中加入100μl1M IPTG以达到1mM的最终浓度(考虑到培养皿中终体积为50ml)。
    2. 加入预热的琼脂(50-55℃)至每管10ml的最终体积;轻轻搅拌。

致谢

这项工作得到墨西哥Consejo Nacional de Ciencia yTecnología(CONACYT;赠款205744和221231)和瓜纳华托大学(赠款936-2016和1090-2016)对M.P-R的支持。急诊室的工作得到了美国国立卫生研究院的资助(R15GM110624)的支持。 KV。 C-C和N.V-N得到了来自CONACYT的奖学金的支持。作者宣称没有利益冲突。

参考

  1. Barajas-Ornelas R del,C.,Ramirez-Guadiana,F. H.,Juarez-Godinez,R.,Ayala-Garcia,V. M.,Robleto,E. A.,Yasbin,R.E。和Pedraza-Reyes,M.(2014)。 PolX对嘌呤/嘧啶类(AP)位点的错误倾向性加工是一种促进适应性的新机制在枯草芽孢杆菌中的诱变。 J Bacter iol 196(16):3012-3022。
  2. Castro-Cerritos,K.V.,Yasbin,R.E.,Robleto,E.A。和Pedraza-Reyes,M.(2017)。核糖核苷酸还原酶在枯草芽孢杆菌胁迫相关的诱变中的作用核糖核苷酸还原酶在枯草芽孢杆菌胁迫相关的诱变中的作用 J> Bacteriol 199(4)。
  3. Gómez-Marroquín,M.,Martin,H.A.,Pepper,A.,Girard,M.E。,Kidman,A.A.,Vallin,C.,Yasbin,R.E.,Pedraza-Reyes,M.and Robleto,E.A。(2016)。 应激枯草芽孢杆菌(Bacillus subtilis)细胞中的固定相诱变通过Mfd依赖(巴塞尔)7(7)。
  4. 5. Kobayashi,K.,Ehrlich,SD,Albertini,A.,Amati,G.,Andersen,KK,Arnaud,M.,Asai,K.,Ashikaga,S.,Aymerich,S.,Bessieres,P Boland,F.,Brignell,SC,Bron,S.,Bunai,K.,Chapuis,J.,Christiansen,LC,Danchin,A.,Debarbouille,M.,Dervyn,E.,Deuerling,E.,我们的研究结果表明,福建省农业科学研究所的研究人员发现, T.,Haga,K.,Harwood,CR,Hecker,M.,Hosoya,D.,Hullo,MF,Kakeshita,H.,Karamata,D.,Kasahara,Y.,Kawamura,F.,Koga,K. ,Koski,P.,Kuwana,R.,Imamura,D.,Ishimaru,M.,Ishikawa,S.,Ishio,I.,Le Coq,D.,Masson,A.,Mauel,C.,Meima,R A.,Moriya,S.,Nagakawa,E.,Nanamiya,H.,Nakai,S.,Nygaard,P.,Ogura,M.,Ohanan,T.,O'Reilly, M.,O'Rourke,M.,Pragai,Z.,Pooley,HM,Rapoport,G.,Rawlins,JP,Rivas,LA,Rivolta,C.,Sadaie,A.,Sadaie,Y.,Sarvas,M Sato,T.,Saxild,HH,Scanlan,E.,Schumann ,W.,Seegers,JF,Sekiguchi,J.,Sekowska,A.,Seror,SJ,Simon,M.,Stragier,P.,Studer,R.,Takamatsu,H.,Tanaka,T.,Takeuchi,M Thomaides,HB,Vagner,V.,van Dijl,JM,Watabe,K.,Wipat,A.,Yamamoto,H.,Yamamoto,M.,Yamamoto,Y.,Yamane,K.,Yata,K. ,Yoshida,K.,Yoshikawa,H.,Zuber,U.和Ogasawara,N.(2003)。 Essential 枯草芽孢杆菌基因。 Proc Natl美国科学院院士 100(8):4678-4683。
  5. Pybus,C.,Pedraza-Reyes,M.,Ross,C.A.,Martin,H.,Ona,K.,Yasbin,R.E。和Robleto,E.(2010)。 压力下的枯草芽孢杆菌细胞中的转录相关突变
  6. Sung,H.M。和Yasbin,R.E。(2002)。 适应性或固定相诱变是枯草芽孢杆菌细菌分化的一个组成部分,。

    J Bacteriol 184(20):5641-5653。

  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Castro-Cerritos, K. V., Villegas-Negrete, N., Ramirez-Ramirez, N., Robleto, E. A. and Pedraza-Reyes, M. (2017). Stationary-phase Mutagenesis Soft-agar Overlay Assays in Bacillus subtilis. Bio-protocol 7(23): e2634. DOI: 10.21769/BioProtoc.2634.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。