活体细胞荧光显微镜观察微生物细胞生长过程中的基本过程
Summary
了解细菌中基本过程的功能是很有挑战性的。荧光显微镜与靶向特定染料可以提供关键的洞察力的微生物细胞生长和细胞周期进展。在这里,农杆菌介导被用作一种模型细菌, 用来强调活细胞成像的方法, 用于描述基本过程。
Abstract
核心细胞过程, 如 DNA 复制和分离, 蛋白质合成, 细胞壁生物合成和细胞分裂依赖于蛋白质的功能, 这是必不可少的细菌生存。一系列的靶向染料可以作为探针, 以更好地理解这些过程。用亲油性染料染色可以观察膜结构、脂微的可视化以及膜泡的检测。使用荧光 d-氨基酸 (FDAAs) 来探测肽生物合成的地点, 可以显示细胞壁生物或细胞生长模式的潜在缺陷。最后, 核酸染色可以显示 DNA 复制或染色体分离可能存在的缺陷。青菁 DNA 染色标签活细胞和适用于定时显微镜, 使 real-time 观察的核形态学在细胞生长。细胞标记协议可应用于蛋白质耗竭突变体, 以识别膜结构、细胞壁生物或染色体分离的缺陷。此外, 延时显微镜可以用来监测形态学变化, 作为一个基本的蛋白质被删除, 并可以提供额外的洞察力的蛋白质功能。例如, 基本细胞分裂蛋白的耗尽导致丝或分支, 而细胞生长蛋白的耗竭可能导致细胞变得更短或更圆。在这里, 细胞生长的协议, 目标特定的标签, 和延时显微镜提供的细菌植物病原体农杆菌介导。同时, 目标特定的染料和延时显微镜能够在A.最后, 所提供的协议可以随时修改, 以探测其他细菌的基本过程。
Introduction
通过细菌细胞周期的进展需要协调许多过程, 包括细胞膜和细胞壁生物合成, DNA 复制和分离, 和细胞分裂。要充分了解细菌细胞生物学的复杂性, 就必须研究这些重要事件;然而, 这是一项不平凡的任务, 因为当这些通路的关键成分被 mutagenized 时, 细胞的生存能力就会受到影响。荧光显微镜结合靶向染料是一个强有力的方法, 以探索这些关键的过程中野生和突变菌株。
肽专用染料包括荧光抗生素 (万古霉素-fl, bocillin) 和荧光 d-氨基酸 (例如, 7-素-3-羧酸 acid-3-amino-d-alanine, 哈达; 4-氯-7-nitrobenzofurazan-3-氨基-d-丙氨酸纳达;tetramethylrhodamine-3-amino-d-alanine;多)。在革兰氏阳性菌中, 使用致死浓度的荧光抗生素类似物来探测肽生物合成的位置是一个有效的策略, 以揭示肽插入模式1,2, 3,4。虽然荧光万古霉素标记已被用来获得洞察肽插入模式固定革兰阴性细菌5, 外膜一般提供了渗透屏障, 防止使用荧光抗生素作为活体细胞肽生物合成的探针。相比之下, 短脉冲的荧光 d-氨基酸或 d-氨基酸与双正交功能基团共价标签地区最近肽插入在广泛的活细菌细胞6,7。用合成 d-氨基酸观察到的肽插入模式包括点状和间隔 (大肠杆菌和枯草芽孢杆菌)、极性和间隔 (农杆菌介导和李斯特菌), 仅间隔 (金黄色葡萄球菌), 和顶端 (链霉菌 venezuelae)6,7。这些观察表明, 细菌表现出不同的细胞壁生物模式, 而使用合成 d-氨基酸作为探针来检测生长模式是许多细菌中的一个有价值的策略。
标记细菌染色体的染料包括脱氧核糖核酸 (脱氧核糖核酸) 具体小槽黏合剂 (46-diamidino-2-吲;DAPI) 和高亲和力菁染料 (绿色和橙色; 见材料清单)。DAPI 染色固定细胞协助计数细菌从环境样品8, 而 DAPI 染色的活细胞被用来表明细菌生存能力9。相比之下, 如橙和绿等菁染料经常被描述为膜 impermeant “死” 细胞污渍, 以枚举不细胞9。值得注意的是, 当这些试剂被用来探测细胞生长过程中细菌核的形态时, DAPI、橙和绿都被证明是膜 permeant, 能够标记活细胞10。在活着的大肠杆菌细胞中, DNA 的 DAPI 染色出现弥漫性, 这是由于荧光从细胞质和 DAPI 染色的细胞重复暴露到紫外线 (UV) 光 perturbs 核结构10。染色大肠杆菌或B. 枯草芽孢杆菌与橙色显示, 这种染料是膜 permeant 和提供持久荧光后, 结合到活细胞 dna, 而不影响细胞生长, dna 复制, 或染色体分离10.这些观察表明, 在许多细菌的细胞生长过程中, 可以使用菁核 DNA 染料来监测其形态。
磷脂特定的 stryl 染料, 如 n-(3-triethylammoniumpropyl)-4-(6-(4-(二乙胺) 苯基) hexatrienyl) 吡啶敌 (4-64; 见材料清单) 是阳离子化合物和副优先与负电荷磷脂, 如心和甘油11。当4-64 被用来标记不同细菌的细胞膜时, 观察到了截然不同的模式。在大肠杆菌中, 4-64 在极点、沿侧壁的带区中以及在晚期 pre-divisional 细胞的分裂部位12中富集。在枯草芽孢杆菌中, 4-64 标记使脂质螺旋的可视化显示为13。在农杆菌介导中, 4-64 标记外膜, 并在一个特征 “马蹄形” 模式中观察到生长极没有标记14,15。这些观察表明, 这些细菌表现出不同的脂质分布, 由于存在的脂肪领域, 导致细胞不对称。4-64 标记模式的变化, 如弥漫标记, 泡或囊泡, 内陷, 或膜萎缩, 可以在特征突变, 影响分布或生物合成的脂质的信息。
除了染色细胞, 确定参与基本过程的蛋白质的功能是必要的。基本蛋白质的定性是技术上的挑战, 因为它是不可能删除的基本基因和研究表型的后果。因此, 出现了消耗蛋白质的替代方法。例如, 一个基本的基因可以被置于一个可诱导的启动子的控制之下, 而不是它的本地启动子。诱导促进剂对小分子反应灵敏, 如;锌16, 异丙基β d-1-thiogalactopyranoside (IPTG)17,18,19,20,21, 糖22, 酸 17,23,和木糖23, 因此靶基因的转录停止, 当诱导剂被移除时, 感兴趣的蛋白质就会耗尽。消耗必需蛋白质的替代方法包括合成开关24 , 它使用小分子 RNA 相互作用阻碍靶基因的转录, CRISPR 干扰25,26阻断靶基因的转录和诱导蛋白的降解27,28 , 它使用肽标签对蛋白质进行 ClpXP 蛋白酶降解。耗尽菌株在细胞丧失生存能力之前只提供了很短的时间来表征, 因此, 在蛋白质耗竭过程中, 细胞的显微成像是一种强有力的表征方法。事实上, 活体细菌细胞显微镜使研究人员能够深入了解基本的生物学过程, 包括细胞形状维护、分泌和条块分割的机制29。
a. 农杆菌是细菌植物病原体30和自然基因工程工程师31,32。因此, 与致病性相关的机制, 包括宿主-病原体相互作用33,34,35, 分泌36, 和主机转换30,31, 37已被广泛调查。要设计防止.. 农杆菌介导的疾病或增强植物转化的策略, 必须更好地了解a. 农杆菌的生存过程。使用目标特定的染料和最近开发的蛋白质耗尽策略. 农杆菌18提供了一种方法来调查基本过程。
在这里, 详细的协议, 显微镜分析的野生, 突变体, 蛋白质枯竭菌株的A. 农杆菌提供。前两个协议描述了如何准备细胞和标签与目标特定的染料。第三个协议为准备琼脂糖垫 (图 1) 和成像细菌细胞 (图 2、 图 3、 图 4) 提供了 step-by 步骤的指导。这些协议也可能适用于其他的细菌与其他的适应, 以考虑不同的媒体条件, 生长速率, 氧要求和细胞结构。
Protocol
Representative Results
Discussion
此协议包含一系列用于调查a. 农杆菌野生、突变体和耗尽菌株的过程。值得注意的是, 《议定书》一节所列的所有程序都可以很容易地适用于其他细菌菌株, 并对培养基、温度和生长速率进行了额外的修改。
使用靶向染料是一个很有价值的工具, 可以为细菌细胞中的细胞周期事件提供详细的描述。在这里, 肽插入模式观察使用荧光 d-氨基酸, 血脂领域是可视化与 4-64, 核被…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢迈克尔 VanNieuwenhze (印第安纳大学) 在图 2和图 4中使用的 FDAAs 的礼物。我们感谢布朗实验室的成员在准备这份手稿时的反馈。研究在褐色实验室在A. 农杆菌细胞成长和分裂由国家科学基金会 (IOS1557806) 支持。
Materials
| Bacterial Strains | |||
| Agrobacterium tumefaciens C58 | ATCC | 33970 | Watson B, Currier TC, Gordon MP, Chilton MD, Nester EW. 1975. Plasmid required for virulence of Agrobacterium tumefaciens. J Bacteriol 123:255-264. |
| Agrobacterium tumefaciens C58ΔtetRA::mini-Tn7T-GM-Ptac-ctrA ΔctrA | Figueroa-Cuilan W, Daniel JJ, Howell M, Sulaiman A, Brown PJB. 2016. Mini-Tn7 insertion in an artificial attTn7 site enables depeltion of the essentail master regulator CtrA in the phytopathogen Agrobacterium tumefaciens. Appl Environ Microbiol. 82:5015-5025. | ||
| Name | Company | Catalog Number | Comments |
| Media Components | |||
| ATGN Minimal Medium | To 1 L of sterilized water add 50 ml 20X Buffer, 50 ml 20X Salts, 12.5 ml 40% glucose. For plates, add 15 g Bacto Agar to 1 L of water and autoclave. Cool to 55 °C and add 50 ml 20X Buffer, 50 ml 20X Salts, 12.5 ml 40% glucose. | ||
| 20X AT Buffer | Add 214 g/L KH2PO4 to water and adjust pH to 7.0 with sodium hydroxide. Autoclave. | ||
| NaOH | Fisher BioReagents | BP359 | |
| KH2PO4 | Fisher Chemical | P288 | |
| 20X AT Salts | Add 40 g/L (NH4)2SO4, 3.2 g/L MgSO4•7H2O, 0.2 g/L CaCl2•2H2O, and 0.024 g/L MnSO4•H2O to water. Autoclave. | ||
| (NH4)2SO4 | Fisher Chemical | A701 | |
| MgSO4•7H2O | Fisher BioReagents | BP213 | |
| CaCl2•2H2O | Fisher BioReagents | BP510 | |
| MnSO4•H2O | Fisher Chemical | M114 | |
| Glucose | Fisher Chemical | D16 | Prepare 40% stock in water. Filter sterilize. |
| Bacto Agar | Fisher BioReagents | BP1423 | Add 15 g to 1 L of water when preparing plates. |
| Name | Company | Catalog Number | Comments |
| Optional Media Additives | |||
| Kanamycin | GoldBio | K-120 | Prepare as a 100 mg/ml stock solution in water and filter sterilize. Use at final concentration of 200 µg/ml. |
| IPTG | GoldBio | I2481C5 | Prepare as a 1 M stock solution in water and filter sterilize. Use at final concentration of 1 mM as needed for induction. |
| Name | Company | Catalog Number | Comments |
| Microscopy Materials | |||
| Microscope Slides | Fisherbrand | 12-550D | 25 X 75 X 1.0 mm. Clean with Sparkle glass cleaner. |
| Microscope Cover Glass | Fisherbrand | 12-541-B | 22 X 22 mm. No. 1.5. Clean with Sparkle glass cleaner. |
| Sparkle Glass Cleaner | Home Depot | 203261385 | Ammonia and alcohol free. |
| Ultra Pure Agarose | Invitrogen | 16500-100 | Add to water, PBS, or media to a final concentration of 1 – 1.5%. Melt in microwave and place on 70 C |
| PBS | Fisher BioReagents | BP399500 | 10X solution to be diluted to 1X with sterile water. |
| Parafilm | Bemis | PM-999 | Laboratory film used as gasket in agarose pad preparation. |
| VALAP | Add equal weights of lanolin, parafin wax, and petroleum jelly to a conical tube. Heat tube in 70 °C dry, bead or water bath to melt and mix. Apply VALAP while still molten. | ||
| Lanolin Butter | SAAQIN | SQ-LAB-R1 | |
| Petroleum Jelly | Target Corp. | 06-17644 | |
| Paraffin Wax | Crafty Candles | 263012 | |
| Name | Company | Catalog Number | Comments |
| Target-specific dyes | |||
| DMSO | Fisher BioReagents | BP231-1 | Use to dilute stock solutions of dyes as needed. |
| FDAAs (NADA, HADA,TADA) | FDAAs can be synthesized or acquired through agreement with Mike VanNieuwenhze (Indiana University). Prepare 100 mM stock solution in DMSO. Use at a final concentration of 5 mM. | ||
| DAPI | ThermoFisher Scientific | 62247 | Prepare 1 mg/ml stock solution in DMSO. Use at final concentration of 1 µg/ml. |
| SYTOX Orange Nucleic Acid Stain | Invitrogen | S11368 | Stock concentration is 5 mM in DMSO. Use at final concentration of 5 µM. |
| FM4-64 | Invitrogen | T3166 | Prepare 8 mg/ml stock solution in DMSO. Use at final concentration of 8 µg/ml. |
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| Dry bath | Sheldon Manufacturing, Inc. | 52120-200 | |
| Metallic thermal beads | Lab Armor | 42370-002 | |
| Epifluorescence microscope equipped with an EMCDD camera | Nikon Eclipse TiE equipped with a QImaging Rolera em-c2 1K electron-multiplying charge-coupled-device (EMCCD) camera is used in this work. |
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