检测皮质片段在细菌孢子萌发
Summary
Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.
Abstract
The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.
Introduction
芽孢是细菌代谢，使细菌在不利的环境中坚持的休眠形式。在许多孢子形成细菌，孢子形成是通过营养剥夺诱导但此过程可通过改变pH值，暴露于氧或其它应力1进行控制。而在其代谢休眠孢子形式，细菌抵抗紫外线辐射，干燥，更高的温度，和冷冻2。最上的孢子形成过程的知识来自在模式生物的研究， 枯草芽孢杆菌 。在孢子形成过程开始于DNA复制和轴向灯丝3,4的形成。非对称隔膜然后把细胞分成两个大小不等车厢。较大的隔间里，母细胞，吞噬较小的隔间里，forespore。无论是母细胞和forespore协调基因表达成熟的孢子母细胞裂解最终，释放多尔曼吨孢子到周围环境中1。
孢子的结构和组合物在许多细菌物种保守的。孢子芯具有比营养细胞低水含量和有丰富的吡啶二羧酸（DPA）1,2。期间形成孢子，DPA被泵入孢子芯换取水。围绕核心的是一种内在的孢子膜的地方，在多数孢子形成的细菌，其中认识到刺激发芽（germinants）的小分子的受体位于2。刚好位于孢子的内膜外是细胞壁肽聚糖的层。一个专门的肽聚糖层（皮层）围绕细胞壁肽聚糖，并且由许多相同的部件，细胞壁肽聚糖[交替的N-乙酰葡糖胺酶（NAG）和N-乙酰胞壁酸（NAM）]的。但是，在皮质的NAM残基的约50％已被转化为胞壁-δ内酰胺5,6- </s了>。期间孢子萌发，这些胞壁-δ内酰胺残基被孢子皮质裂解酶（SCLEs）的认可，因此允许皮质被降解但不细胞壁（必需完成发芽过程）。周围皮质的外膜和外壳蛋白2几层。
控制发芽的方法对于形成孢子的细菌至关重要。当开端受体各自germinants 2交互萌发开始。在许多孢子形成的细菌，这些germinants是氨基酸，糖，核苷酸，离子或它们的2组合。C.难辨孢子萌发是由某些胆汁酸，[ 例如 ，牛磺胆酸（TA）]和氨基酸（ 例如 ，甘氨酸）7-10的组合引发。虽然有在C之间的差异难辨发芽途径和在所研究的途径其他孢子形成细菌，如B.枯草芽孢杆菌，共同所有的降解孢子皮质以允许营养细胞以从发芽孢子2,8成长的绝对要求。皮质降解可由SCLEs CwlJ / SleB来实现或SLEC（如在枯草芽孢杆菌中发现）（如在许多梭菌找到）。皮质水解降低了细胞壁和孢子的约束。这允许充分芯补液，在重新激活许多必需的细胞代谢2蛋白质的一个必要步骤。
当孢子发芽，从相亮状态到相暗状态，这个过程的休眠孢子的变化可以通过在600光密度（OD）的变化来测量 纳米11。先前的一份报告表明，在很多OD这一变化是由于DPA从12孢子释放。在我们最近的研究中，我们试图比较C的定时难辨孢子萌发和监测DPA和皮质碎片9的释放。在这项研究中是至关重要的监测皮质片段的释放，他们开始由萌芽孢子被释放。
此处所使用的比色测 定法是基于与还原末端检测糖的方法开发Ghuysen 等 13。因为其他人描述协议检测还原糖14或修改这些协议15，关于这一问题的文献可能会造成混淆。在这里，我们详细地一步一步方法还原糖的比色检测从发芽C.解放艰难梭菌芽孢。虽然本研究采用C.艰难梭菌芽孢，从其他孢子形成的细菌孢子的发芽过程中释放的还原糖能够与该协议9,16,17进行检测。
Protocol
Representative Results
Discussion
在刺激时，进行发芽的过程孢子丧失其抗损伤性，而无需大分子合成。当孢子萌发被触发时，孢子释放核心DPA，以换取水2。由于休眠孢子的高DPA含量，孢子核心是在强烈渗透压和专门肽，皮质，有助于防止芯从通过作用，据推测，作为屏障，以扩张2肿胀。
上述的方法使用艾氏试剂（黄色溶液），以检测还原糖作为可测量的移位到紫色色调的存在。期间皮质?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
描述该项目由奖号5R01AI116895从过敏和传染病国家研究所的支持。内容完全是作者的责任，并不一定代表国家过敏和传染病研究所和美国国立卫生研究院的官方意见。
Materials
| 2.0 mL screw cap tube | USA scientific | 1420-3700 | |
| p1000 eppendorf pippet | Eppendorf | ||
| p200 eppendorf pippet | Eppendorf | ||
| p20 eppendorf pippet | Eppendorf | ||
| 100-1250uL pipet tips | VWR | 89079-486 | |
| 1-200uL pipet tips | VWR | 89079-458 | |
| N-acetyl-D-glucosamine | Sigma | A3286-25G | |
| Hydrochloric Acid – 10N | BDH-Aristar | BDH3032-3.8LP | |
| Acetic Anhydride | Alfa Aesar | L04295 | |
| Sodium Bicarbonate | BDH | BDH0280-500G | |
| Potassium Tetraborate tetrahydrate | Alfa Aesar | 39435 | |
| Sodium Hydroxide | BDH | BDH8019-500G | |
| 4-(Dimethylamino)benzaldehyde | Sigma | 156477-100G | |
| Saturated Phenol | Fisher | BP1750-400 | |
| 2-Mercaptoethanol | Aldrich | M6250-100ML | |
| SpectraMax M3 | Molecular Devices | ||
| Acetic Acid, Glacial | BDH | BDH3098-3.8LP | |
| Heated, Circulating Water Bath | VWR Scientific | Model 1136 | |
| Microtest 96 | Falcon | 353072 | 96 well clear tissue culture plates |
| Culture tubes | VWR | 89000-506 | |
| Lyophilizer | |||
| Heat Blocks | |||
| Vortex Machine | |||
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