通过共生荧光标记菌株监测细菌细胞群中的物种内部竞争
Summary
细菌在有生之年可能会积累有害或有益的突变。在积累有益突变的细胞群中，个体可能迅速比其研究员竞争。在这里，我们介绍了一个简单的程序，以可视化物种内部竞争的细菌细胞种群随着时间的推移使用荧光标记的个人。
Abstract
许多微生物，如细菌的增殖速度极快，种群可能达到高细胞密度。种群中很小一部分细胞总是积累的突变，这些突变对细胞有害或有益。如果突变的健身效应为亚人口提供了强大的选择性生长优势，这种亚种群的个体可能会迅速竞争，甚至完全消除其直接研究员。因此，小的基因变化和获得有益突变的细胞的选择驱动积累可能导致细胞种群的基因型完全改变。在这里，我们提出了一个程序，监测快速克隆扩张和消除有益和有害的突变，分别在细菌细胞群随着时间的推移，通过共生荧光标记个体的Gram阳性模型杆菌 亚蒂利斯。该方法易于执行，非常能说明细菌细胞群中个体之间的物种间竞争。
Introduction
土壤细菌通常具有灵活的调控网络和广泛的代谢能力。这两个特征使细胞能够调整其催化物和合成代谢途径，以与研究员和其他微生物竞争营养物质，这些营养物质在给定的生态利基1中可用。然而，如果细菌不能适应其环境，其他机制可能决定一个物种的生存。事实上，由于许多细菌迅速增殖，种群可以达到高细胞密度亚种群，可能自发地积累了有益的突变，为细胞提供了选择性的生长优势，从而增加了细胞的适应能力。此外，突变热点和压力引起的适应性突变可以促进一种不适应细菌2，3的进化。因此，在连续选择下突变的积累和生长是巨大的微生物多样性的起源，即使在同一属4，5。与自然界一样，由于在选择下不断培养，细菌基因组的形成也会在实验室中发生。Gram阳性细菌B.亚提利的驯化就是例证，该细菌在全世界用于基础研究和工业。在20世纪40年代，B.亚提利斯接受了DNA损伤X射线治疗，随后在特定的生长条件下进行了培养。细菌在驯化过程中积累的突变导致许多生长特征的丧失，即B.亚提利实验室菌株168丧失了形成复杂菌落的能力7，8。
如今，对于研究最好的大肠杆菌和B.亚提利菌模型，各种强大的工具可用于基因操纵其基因组，以解决特定的科学问题。有时，兴趣基因的失活会导致严重的生长缺陷，然后在标准生长介质9上清晰可见。相比之下，导致生长缺陷微弱，从而只轻微影响菌株适应的突变往往被忽视。然而，在这两种情况下，长期潜伏和传递的突变菌株几代人通常会导致抑制突变体的积累，已经恢复了表型的父菌株2，9。抑制突变体的特征和突变的识别，已经恢复了母突变株的生长缺陷是一个非常有用的方法，允许阐明重要和往往新颖的细胞过程10，11。
我们有兴趣控制谷氨酸平衡在B.亚提利斯12。与大肠杆菌类似，B. 亚提利通过抑制突变体的积累对谷氨酸平衡（即谷氨酸降解2中的块）的扰动做出反应。这些自发突变获得的抑制突变体的基因组变化被证明可以迅速恢复谷氨酸平衡9，13。因此，在细菌驯化过程中，B.亚提利适应特定生长状况，反映在酶合成和进化的酶活性中，这不足为奇，这些活动涉及谷氨酸代谢12。有人提出，在驯化过程中生长介质中缺乏外源性谷氨酸，是实验室菌株1682，14中神秘谷氨酸脱氢酶（GDH）gudB CR基因出现和固定的驱动力。这一假设得到我们观察的支持，即实验室菌株中GDH活性的减少为细菌提供了选择性生长优势，而外源性谷氨酸是稀缺的2。此外，在没有外源性谷氨酸的情况下，培育B.亚提利菌株，合成GDH GudB，导致抑制突变体的积累，这些突变体已经灭活了gadB基因2。显然，阴性活性GDH的存在对细胞不利，因为内源性产生的谷氨酸，否则可用于合成，会降解为铵和2-牛油酸盐（图1）。相比之下，当谷氨酸由介质提供时，配备高级 GDH 活性的B. 亚提利菌株比只合成一种功能性 GDH 的菌株具有选择性生长优势。有理由假设，高GDH活性允许细菌利用谷氨酸作为第二碳源，除了介质2提供的其他碳源（见图1）。因此，GDH 活性强烈地影响细菌的健身，具体取决于外源性谷氨酸的供应情况。
在这里，我们提出了一个非常说明性的方法来监测和可视化两个 B.亚蒂利斯 菌株之间的物种间竞争，在染色体上的单个轨迹不同（图2）。这两种菌株被标记为编码氟磷YFP和CFP的 yfp 和cfp的yfp和 cfp 基因，并在不同的营养条件下共同培养。通过随着时间的推移采样，并通过在阿加板块上电镀适当的稀释，每个培养物中的幸存者可以使用常见的立体荧光显微镜轻松监测。本文描述的程序是容易执行和适合可视化快速克隆扩张和消除有益和有害的突变，分别在细胞群随着时间的推移。
Protocol
Representative Results
Discussion
已经开发了几种方法来分析细菌16的竞争性健康。在许多情况下，细菌被标记为不同的抗生素耐药性盒17。与我们的方法类似，用抗生素耐药盒标记细胞可以评估在规定的生长条件下共生过程中细菌的竞争性健康。此外，这种方法可用于确定17号染色体上特定轨迹中彼此不同的细胞的竞争性适应性。然而，使用抗生素耐药盒来监测竞技体能存在一些缺点。由于耐药基因的表?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者实验室的工作得到了德国福雄格梅因沙夫特（http://www.dfg.de） 的支持;CO 1139/1-1）、化学工业基金会（http://www.vci.de/fonds）和哥廷根分子生物学中心（GZMB）。作者要感谢约尔格·斯图尔克对手稿的有益评论和批判性解读。
Materials
| (NH4)2SO4 | Roth, Germany | 3746 | – |
| Agar | Difco, USA | 214010 | – |
| Ammonium ferric citrate (CAF) | Sigma-Aldrich, Germany | 9714 | – |
| CaCl2 | Roth, Germany | 5239 | – |
| Glucose | Applichem, Germany | A3617 | – |
| Glycerol | Roth, Germany | 4043 | – |
| K2HPO4 x 3 H2O | Roth, Germany | 6878 | – |
| KCl | Applichem, Germany | A3582 | – |
| KH2PO4 | Roth, Germany | 3904 | – |
| KOH | Roth, Germany | 6751 | – |
| MgSO4 x 7 H2O | Roth, Germany | P027 | – |
| MnCl2 | Roth, Germany | T881 | – |
| MnSO4 x 4 H2O | Merck Millipore, Germany | 102786 | – |
| NaCl | Roth, Germany | 9265 | – |
| Nutrient broth | Roth, Germany | X929 | – |
| Potassium glutamate | Applichem, Germany | A3712 | – |
| Tryptone | Roth, Germany | 8952 | – |
| Tryptophan | Applichem, Germany | A3445 | – |
| Yeast extract | Roth, Germany | 2363 | – |
| 1.5 ml Reaction tubes | Sarstedt, Germany | 72,690,001 | – |
| 2.0 ml Reaction tubes | Sarstedt, Germany | 72,691 | – |
| 15 ml Plastic tubes with screw cap | Sarstedt, Germany | 62,554,001 | – |
| Petri dishes | Sarstedt, Germany | 82.1473 | – |
| 1.5 ml Polystyrene cuvettes | Sarstedt, Germany | 67,742 | – |
| 15 ml Glass culture tubes | Brand, Germany | 7790 22 | – |
| with aluminium caps | |||
| 100 ml Shake flasks with aluminium caps | Brand, Germany | 928 24 | – |
| Sterile 10 ml glass pipettes | Brand, Germany | 278 23 | – |
| Incubator (28 and 37 °C) | New Brunswick | M1282-0012 | – |
| Standard pipette set (2-20 μl, 10-100 μl, 100-1000 μl) | Eppendorf, Germany | 4910 000.034, 4910 000.042, | – |
| 4910 000.042, | |||
| 4910 000.069 | |||
| Table top centrifuge for 1.5 and 2 ml reaction tubes | Thermo Scientific, Heraeus Fresco 21, Germany | 75002425 | – |
| Table top centrifuge for 15 ml plastic tubes | Heraeus Biofuge Primo R, Germany | 75005440 | – |
| Standard spectrophotometer | Amersham Biosciences Ultrospec 2100 pro, Germany | 80-2112-21 | – |
| Stereofluorescence microscope | Zeiss SteREO Lumar V12, Germany | 495008-0009-000 | – |
| Freezer (-20 and -80 °C) | – | – | – |
| Fridge (4 °C) | – | – | – |
| Autoclave | Zirbus, LTA 2x3x4, Germany | – | – |
| pH meter | pH-meter 766, Calimatic, Knick, Germany | 766 | – |
| Vortex | Vortex 3, IKA, Germany | 3340000 | – |
| Balance | CP2202S, Sartorius, Germany | replaced by | – |
| CPA2202S | |||
| Black pen (permanent marker) | Staedler, Germany | 317-9 | – |
| Powerpoint program | Microsoft, USA | – | – |
| Office Excel program | Microsoft, USA | – | Program for data processing |
| Adobe Photoshop CS5 | Adobe, USA | replaced by CS6, download | Computer program for image processing |
| Computer | PC or Mac | – | – |
| ZEN pro 2011 software for the stereofluorescence microscope | Zeiss, Germany | 410135 1002 110 | AxioCam MRc Rev. Obtained through Zeiss |
| Specific solution recipes | |||
| SP medium | |||
| 8 g Nutrient broth | |||
| 0.25 mg MgSO4 x 7 H2O | |||
| 1 g KCl | |||
| if required, add 15 g agar for solid SP medium | |||
| ad 1 l with H2O, autoclave for 20 min at 121 °C | |||
| 1 ml CaCl2 (0.5 M), sterilized by filtration | |||
| 1 ml MnCl2 (10 mM) sterilized by filtration | |||
| 2 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration | |||
| LB medium | |||
| 10 g Tryptone | |||
| 5 g Yeast extract | |||
| 10 g NaCl | |||
| if required, add 15 g agar for solid LB medium | |||
| ad 1 l with H2O, autoclave for 20 min at 121 °C | |||
| C-Glc minimal medium | |||
| 200 ml 5 x C salts | |||
| 10 ml L-Tryptophan (5 mg/ml), sterilized by filtration | |||
| 10 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration | |||
| 10 ml III’ salts | |||
| 25 ml Glucose (20%), autoclaved for 20 min at 121 °C | |||
| ad 1 l with sterile H2O | |||
| CE-Glc minimal medium | |||
| 200 ml 5 x C salts | |||
| 10 ml L-Tryptophan (5 mg/ml), sterilized by filtration | |||
| 10 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration | |||
| 10 ml III’ salts | |||
| 20 ml Glutamate (40%) | |||
| 25 ml Glucose (20%), autoclaved for 20 min at 121 °C | |||
| ad 1 l with sterile H2O | |||
| 5 x C salts | |||
| 20 g KH2PO4 | |||
| 80 g K2HPO4 x 3 H2O | |||
| 16.5 g (NH4)2SO4 | |||
| ad 1 l with sterile H2O, autoclave for 20 min at 121 °C | |||
| III’ salts | |||
| 0.232 g MnSO4 x 4 H2O | |||
| 12.3 g MgSO4 x 7 H2O | |||
| ad 1 l with sterile H2O, autoclave for 20 min at 121 °C | |||
| 40% Glutamate solution | |||
| 200 g L-Glutamic acid | |||
| adjust the pH to 7.0 by adding approximately 80 g KOH | |||
| ad 0.5 l with sterile H2O, autoclave for 20 min at 121 °C | |||
| 0.9% Saline (NaCl) solution | |||
| ad 1 l with sterile H2O, autoclave for 20 min at 121 °C | |||
| 50% Glycerol solution | |||
| 295 ml Glycerol (87%) | |||
| ad 0.5 l with sterile H2O, autoclave for 20 min at 121 °C | |||
| Bacteria (All strains are based on the Bacillus subtilis strain 168) | |||
| Bacillus subtilis BP40 (rocG+ gudBCR amyE::PgudB-yfp) | Laboratory strain collection | ||
| Bacillus subtilis BP41 (rocG+ gudBCR amyE::PgudB-cfp) | |||
| Bacillus subtilis BP52 (rocG+ gudB+ amyE::PgudB-cfp) | |||
| Bacillus subtilis BP156 (rocG+ gudB+ amyE::PgudB-yfp) | |||
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